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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6841—In situ hybridisation
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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing
  • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
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  • C12Y—ENZYMES
  • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
  • C12Y207/07—Nucleotidyltransferases (2.7.7)
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  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2458/00—Labels used in chemical analysis of biological material
  • G01N2458/10—Oligonucleotides as tagging agents for labelling antibodies

Definitions

• Cells within a tissue of a subject have differences in cell morphology and/or function due to varied analyte levels (e.g., gene and/or protein expression) within the different cells.
• the specific position of a cell within a tissue e.g., the cell's position relative to neighboring cells or the cell's position relative to the tissue microenvironment
• Chromatin structure can be different between cells in a biological sample or between biological samples from the same tissue. Assaying differences in accessible chromatin can be indicative of transcriptionally active sequences, e.g., genes, in a particular cell. Further understanding the transcriptionally active regions within chromatin will enable identification of which genes contribute to a cell's function and/or phenotype.
• the present disclosure generally describes methods for spatially analyzing genomic DNA present in a biological sample.
• the method comprises providing an array with a plurality of capture probes such that a capture probe of the plurality comprises a spatial barcode and a capture domain; permeabilizing the biological sample under conditions sufficient to make the genomic DNA in the biological sample accessible to a transposon insertion; providing a transposon sequence and a transposase enzyme to the biological sample under conditions wherein the transposon sequence is inserted into the genomic DNA; allowing the transposase enzyme to excise the inserted transposon sequence from the genomic DNA thus generating fragmented genomic DNA; contacting the biological sample comprising the fragmented genomic DNA with an array under conditions such that a capture probe interacts with the fragmented genomic DNA; and correlating the location of the capture probe on the array to a location in the biological sample, thereby spatially analyzing the fragmented genomic DNA.
• the array comprising a plurality of capture probes are provided on a substrate. In some embodiments, the array comprising the plurality of capture probes is provided on a feature. In some embodiments, the capture probe is directly or indirectly attached. In some embodiments, the array comprising the plurality of capture probes is provided on the feature on the substrate. In some embodiments, the substrate comprises a microfluidic channel. In some embodiments, the capture probe further comprises one or more of a cleavage domain, a functional domain, and a unique identifier, or combinations thereof.
• a further migration step comprising a step wherein the fragmented genomic DNA is migrated to the substrate.
• the migration step is an active migration step comprising applying an electric field to the fragmented genomic DNA.
• the migration step is a passive migration step comprising diffusion.
• the migration of the fragmented genomic DNA from the biological sample comprises exposing the biological sample and the feature to heat.
• the biological sample is immobilized on the substrate.
• the transposase enzyme is a dimer comprised of a first monomer complexed with a first adapter comprising a transposon end sequence and a sequence complementary to the capture domain and wherein a second monomer is complexed with a second adapter comprising a transposon end sequence and a second adapter sequence, wherein the transposase enzyme ligates the first adapter and the second adapter to the fragmented genomic DNA.
• the first adapter and the second adapter have a 5′ end and a 3′ end, wherein the 5′ end is phosphorylated in situ.
• the 5′ end of the first adapter complexed with the first monomer and the second adapter complexed with the second monomer are phosphorylated.
• the step of phosphorylating the 5′ end of the first adapter complexed with the first monomer and the second adapter complexed with the second monomer comprises contacting a first monomer:first adapter complex and a second monomer:second adapter complex with a polynucleotide kinase in the presence of ATP.
• the capture domain of the capture probe comprises a sequence that hybridizes to the sequence complementary to the capture domain of the first adapter.
• the capture probe is a partially double stranded molecule comprising a first strand comprising the capture domain hybridized to a second strand, and wherein the first strand templates the ligation of the first adapter to the second strand.
• the first adapter sequence complementary to the capture domain, or portion thereof, hybridized to the capture probe templates the ligation and ligating the 5′ end of the first adapter to the 3′ end of the capture probe.
• the capture probe comprises a surface probe and a splint oligonucleotide and the splint oligonucleotide comprises a sequence complementary to a hybridization domain of the surface probe.
• the splint oligonucleotide comprises the capture domain with a sequence complementary to the first adapter, or portion thereof.
• the splint oligonucleotide hybridizes to the first adapter, or portion thereof, and to the hybridization domain of the surface probe, or portion thereof.
• ligation is performed in the presence of the splint oligonucleotide, thereby ligating the surface probe of the capture probe and the first adapter.
• the fragmented genomic DNA hybridized to the capture probe by the first adapter is an extension template used to produce an extended capture probe that comprises the sequences of the spatial barcode and a sequence complementary to the fragmented genomic DNA.
• the capture probe hybridized to the fragmented genomic DNA is extended with a DNA polymerase.
• the DNA polymerase has strand displacement activity.
• a further step of gap repair of single stranded breaks in the fragmented genomic DNA.
• the sequence complementary to the capture domain is a unique sequence.
• the capture probe is ligated to the fragmented genomic DNA by a DNA ligase enzyme.
• the transposase enzyme is a Tn5 transposase, or a functional derivative thereof.
• the Tn5 transposase enzyme comprises a sequence having at least 80% identity to SEQ ID NO: 1.
• the transposase enzyme is a Mu transposase, or the functional derivative thereof.
• the Mu transposase enzyme comprises a sequence having at least 80% identity to SEQ ID NO: 2.
• the transposon end sequence comprises a sequence having at least 80% identity to SEQ ID NO. 8.
• the transposon end sequence comprises a sequence having at least 80% identity to any one of SEQ ID NO: 9 to 14.
• permeabilizing the biological sample is performed under a chemical permeabilization condition, an enzymatic permeabilization condition, or both.
• the chemical permeabilization condition comprises contacting the biological sample with an alkaline solution.
• the enzymatic permeabilization condition comprises contacting the biological sample with an acidic solution comprising a protease enzyme.
• the protease enzyme is an aspartyl protease, preferably a pepsin enzyme, a pepsin-like enzyme, or the functional equivalent thereof.
• the pepsin enzyme, the pepsin-like enzyme, or the functional equivalent thereof comprises a sequence having at least 80% identity to SEQ ID NO: 3 or 4.
• the enzymatic permeabilization condition comprises contacting the biological sample with a zinc endopeptidase, a collagenase enzyme, a collagenase-like enzyme, or a functional equivalent thereof; a serine protease, a proteinase K enzyme, a proteinase K-like enzyme, or a functional equivalent thereof, or both.
• the collagenase enzyme, the collagenase-like enzyme, or the functional equivalent thereof comprises a sequence having at least 80% identity to SEQ ID NO: 5 or 6.
• the proteinase K enzyme, the proteinase K-like enzyme, or the functional equivalent thereof comprises a sequence having at least 80% identity to SEQ ID NO: 7.
• the fragmented genomic DNA hybridized to the capture probe as the extension template generates a DNA molecule.
• the fragmented genomic DNA hybridized to the capture probe acts as a ligation template to generate a DNA molecule.
• the step of analyzing the DNA molecule includes sequencing.
• the step of correlating the spatial barcode of the capture probe with the fragmented genomic DNA associated with the capture probe spatially analyzes the fragmented genomic DNA.
• the biological sample is imaged before or after contacting the biological sample with the substrate.
• the present disclosure generally describes a kit for use in a method of spatially detecting nucleic acids of a biological sample, wherein the kit comprises any two or more of an array on which plurality of capture probes are present; one or more biological sample permeabilization reagents; one or more transposase enzymes; one or more reverse transcriptases; and one or more cleavage enzymes.
• the present disclosure generally describes a method for spatial analysis of genomic DNA and RNA present in a biological sample wherein an array is provided and the array comprises a plurality of capture probes, wherein a first capture probe of the plurality of capture probes comprises a spatial barcode and a first capture domain, and wherein a second capture probe of the plurality of capture probes comprises the spatial barcode and a second capture domain; permeabilizing the biological sample under conditions sufficient to make the genomic DNA in the biological sample accessible to transposon insertion; providing a transposon sequence and a transposase enzyme to the biological sample under conditions wherein the transposon sequence is inserted into the genomic DNA;
• the transposase enzyme to excise the inserted transposon sequence from the genomic DNA, thereby generating fragmented genomic DNA; contacting the biological sample comprising the fragmented genomic DNA and RNA with the array under conditions where the first capture domain interacts with the fragmented genomic DNA and the second capture domain interacts with the RNA; and correlating the location of the first capture probe on the array to a location in the biological sample and correlating the location of the second capture probe on the array to a location in the biological sample, thereby spatially analyzing the fragmented genomic DNA and RNA at the location in the biological sample.
• the RNA is a mRNA.
• the first capture domain and the second capture domain are identical.
• the first capture domain and the second capture domain comprise a homopolymeric poly (T) sequence.
• the first capture domain and the second capture domain are different.
• the first capture domain comprises a random sequence and the second capture domain comprises a poly (T) sequence.
• the array comprising the plurality of capture probes is provided on a substrate.
• the array comprising the plurality of capture probes is provided on a feature.
• the feature comprises the first capture probe, the second capture probe, or both.
• the first capture probe, the second capture probe, or both are directly or indirectly attached.
• the array comprising the plurality of capture probes is provided on the feature on the substrate.
• the substrate comprises a microfluidic channel.
• the first capture probe, the second capture probe, or both comprise one or more of a cleavage domain, a functional domain, and a unique identifier, or combinations thereof.
• the migration step there is a migration step wherein the fragmented genomic DNA and the RNA are migrated to the substrate.
• the migration step is an active migration step.
• the migration step is a passive migration step.
• the migration of the fragmented genomic DNA and the RNA from the biological sample comprises exposing the biological sample to heat.
• the biological sample is immobilized on the substrate.
• the fragmented genomic DNA is repaired by ligating breaks with a ligase enzyme. In some embodiments, single stranded breaks in the fragmented genomic DNA undergo gap repair.
• a sequence complementary to the first capture domain of the first capture probe is introduced to the fragmented genomic DNA. In some embodiments, the first capture domain of the first capture probe hybridizes to the sequence complementary to the capture domain introduced to the fragmented genomic DNA. In some embodiments, the random sequence of the first capture domain hybridizes the fragmented genomic DNA.
• the second capture domain of the second capture probe hybridizes to a complementary sequence in the mRNA. In some embodiments, the sequence complementary to the first capture domain and the complementary sequence in the mRNA is a homopolymeric sequence. In some embodiments, the homopolymeric sequence is a poly(A) sequence.
• extension of the first capture probe using the fragmented genomic DNA as an extension template, and extension of the second capture probe using the RNA as an extension template is performed.
• extending the first capture probe is performed with a DNA polymerase.
• extending the second capture probe is performed with reverse transcriptase.
• transposase is a Tn5 transposase, or a functional derivative thereof.
• the Tn5 transposase enzyme comprises a sequence having at least 80% identity to SEQ ID NO: 1.
• the transposase enzyme is a Mu transposase enzyme, or a functional derivative thereof.
• the Mu transposase enzyme comprises a sequence having at least 80% identity to SEQ ID NO: 2.
• the transposase enzyme is complexed with an adapter comprising a transposon end sequence.
• the transposon end sequence comprises a sequence having at least 80% identity to SEQ ID NO: 8.
• the transposon end sequence comprises a sequence having at least 80% identity to any one of SEQ ID NO: 9 to 14.
• a step of permeabilizing the biological sample is performed.
• the chemical permeabilization condition comprises contacting the biological sample with an alkaline solution.
• the enzymatic permeabilization condition comprises contacting the biological sample with an acidic solution comprising a protease enzyme.
• the protease enzyme is an aspartyl protease, preferably a pepsin enzyme, a pepsin-like enzyme, or a functional equivalent thereof.
• the pepsin enzyme, the pepsin-like enzyme, or functional equivalent thereof comprises a sequence having at least 80% identity to SEQ ID NO: 3 or 4.
• the enzymatic permeabilization condition comprises contacting the biological sample with a zinc endopeptidase, a collagenase enzyme, a collagenase-like enzyme, or a functional equivalent thereof, a serine protease, a proteinase K enzyme, a proteinase K-like enzyme, or a functional equivalent thereof, or both.
• the collagenase enzyme, the collagenase-like enzyme, or the functional equivalent thereof comprises a sequence having at least 80% identity to SEQ ID NO: 5 or 6.
• the proteinase K enzyme, the proteinase K-like enzyme, or the functional equivalent thereof comprises a sequence having at least 80% identity to SEQ ID NO: 7.
• step of analyzing the DNA molecule includes sequencing. In some embodiments, correlating the spatial barcode of the first capture probe with the fragmented genomic DNA associated with the first capture probe spatially analyzes the fragmented genomic DNA. In some embodiments, correlating the spatial barcode of the second capture probe with the mRNA associated with the second capture probe spatially analyzes the mRNA. In some embodiments, the biological sample is imaged before or after contacting the biological sample with the substrate.
• each when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.
• FIG. 1 shows an exemplary spatial analysis workflow.
• FIG. 2 shows an exemplary spatial analysis workflow.
• FIG. 3 shows an exemplary spatial analysis workflow.
• FIG. 4 shows an exemplary spatial analysis workflow.
• FIG. 5 shows an exemplary spatial analysis workflow.
• FIG. 6 is a schematic diagram showing an example of a barcoded capture probe, as described herein.
• FIG. 7 is a schematic illustrating a cleavable capture probe, wherein the cleaved capture probe can enter into a non-permeabilized cell and bind to target analytes within the sample.
• FIG. 8 is a schematic diagram of an exemplary multiplexed spatially-labelled feature.
• FIG. 9 is a schematic diagram of an exemplary analyte capture agent.
• FIG. 10 is a schematic diagram depicting an exemplary interaction between a feature-immobilized capture probe 1024 and an analyte capture agent 1026 .
• FIGS. 11 A, 11 B, and 11 C are schematics illustrating how streptavidin cell tags can be utilized in an array-based system to produce a spatially-barcoded cells or cellular contents.
• FIG. 12 is a schematic showing the arrangement of barcoded features within an array.
• FIG. 13 is a schematic illustrating a side view of a diffusion-resistant medium, e.g., a lid.
• FIGS. 14 A and 14 B are schematics illustrating expanded FIG. 14 A and side views FIG. 14 B of an electrophoretic transfer system configured to direct transcript analytes toward a spatially-barcoded capture probe array.
• FIGS. 15 A-G is a schematic illustrating an exemplary workflow protocol utilizing an electrophoretic transfer system.
• FIG. 16 shows an example of a microfluidic channel structure 1600 for partitioning dissociated sample (e.g. biological particles or individual cells from a sample).
• dissociated sample e.g. biological particles or individual cells from a sample.
• FIG. 17 A shows an example of a microfluidic channel structure 1700 for delivering spatial barcode carrying beads to droplets.
• FIG. 17 B shows a cross-section view of another example of a microfluidic channel structure 1750 with a geometric feature for controlled partitioning.
• FIG. 17 C shows a workflow schematic
• FIG. 18 is a schematic depicting cell tagging using either covalent conjugation of the analyte binding moiety to the cell surface or non-covalent interactions with cell membrane elements.
• FIG. 19 is a schematic depicting cell tagging using either cell-penetrating peptides or delivery systems.
• FIG. 20 A is a workflow schematic illustrating exemplary, non-limiting, non-exhaustive steps for “pixelating” a sample, wherein the sample is cut, stamped, microdissected, or transferred by hollow-needle or microneedle, moving a small portion of the sample into an individual partition or well.
• FIG. 20 B is a schematic depicting multi-needle pixilation, wherein an array of needles punched through a sample on a scaffold and into nanowells containing gel beads and reagents below. Once the needle is in the nanowell, the cell(s) are ejected.
• FIG. 21 shows a workflow schematic illustrating exemplary, non-limiting, non-exhaustive steps for dissociating a spatially-barcoded sample for analysis via droplet or flow cell analysis methods.
• FIGS. 22 A-D is a schematic diagram showing an example of spatially processing DNA from a biological sample.
• FIGS. 23 A-C is a schematic diagram showing an example of a spatial ATAC-seq method.
• FIGS. 24 A-C is a schematic diagram showing an example of multiplex detection of analytes in a biological sample.
• FIG. 25 is a schematic diagram showing a representative workflow of the invention.
• FIG. 26 is a schematic diagram showing a representative workflow of the procedure used to investigate Tn5 transposase/transposome efficiency.
• FIG. 27 is a schematic diagram showing a representative workflow of the procedure used to investigate tagmentation conditions in immobilized tissue sections.
• FIG. 28 is a schematic diagram showing a representative workflow of the procedure used to investigate hybridization and ligation conditions of phosphorylated DNA tagments.
• FIG. 29 shows DNA fragment analysis of a reference tagmentation reaction performed in a cellular suspension as described (Corces, M. R., et. al., Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution, Nat Genetic . vol. 48(10): pp. 1193-1203 (2016)). Fragment distribution analysis is used to determine the success of open chromatin tagmentation, wherein a successful tagmentation reaction of accessible chromatin reveals a periodicity (approx. 170-180 bp; nucleosome-wrapped DNA and PCR handles) in the size of PCR-amplified nucleosome-protected DNA fragments.
• FIGS. 30 A-E shows a DNA fragment analysis of tagmentation reactions performed according to the workflow in FIG. 27 comparing different detergents in the permeabilization step performed for 10 minutes at 25° C.: FIG. 30 A ) no detergent; FIG. 30 B ) 0.1% Triton-X-100; FIG. 30 C ) IGEPAL 0.1%; FIG. 30 D ) Tween 0.1%, Digitonin 0.01% and NP-40 0.1%.
• insert size distribution analysis on a tissue section permeabilized with IGEBAL 0.1% and processed as in (Chen 2016 Nat Meth) fails to reveal a prominent nucleosome periodicity.
• FIGS. 31 A-D shows a DNA fragment analysis of tagmentation reactions performed according to the workflow in FIG. 27 comparing different protease treatments (3 minutes) on an immobilized tissue section:
• FIG. 31 A Pepsin (0.1 mg/ml) in presence of 100 mM HCL;
• FIG. 31 B Pepsin (0.5 mg/ml) in the presence of 0.5M acetic acid;
• FIG. 31 C Pepsin (0.1 mg/ml) in the presence of 0.5M acetic acid;
• FIG. 31 D Proteinase K.
• FIGS. 32 A-C shows a DNA fragment analysis of tagmentation reactions performed according to the workflow in FIG. 27 comparing different permeabilization treatments on an immobilized tissue section: FIG. 32 A ) Pepsin (0.1 mg/ml) in the presence of 0.5 acetic acid;
• FIG. 32 B chemical permeabilization using 1 ⁇ Exonuclease-I buffer (67 mM Glycine-KOH, 6.7 mM MgCl 2 , 10 mM ⁇ -ME); and FIG. 32 C ) Collagenase.
• FIGS. 33 A-C shows a DNA fragment analysis of tagmentation reactions performed according to the workflow in FIG. 27 comparing different Tn5 assembly methods on an immobilized tissue section:
• FIG. 33 A MEDS-Tn5 assembled on column as in (Picelli, S., et. al., Tn5 transposase and tagmentation procedures for massively scaled sequencing projects; Genome Res ., vol. 24, 2033-2040 (2014));
• FIG. 33 B MEDS-Tn5 assembled in solution as in (Picelli et al., 2014, supra);
• FIG. 33 C MEDS-Tn5 assembly with 5′ phosphorylated oligonucleotides assembled in solution.
• FIG. 34 is a schematic diagram showing a representation of the tests to assess the effect of post-assembly T4-PNK phosphorylation and reaction conditions on MEDS Tn5 complexes.
• FIGS. 35 A-D shows a DNA fragment analysis of tagmentation reactions performed according the workflow in FIG. 26 investigating the compatibility of post-assembly 5′ phosphorylation with DNA tagmentation
• FIG. 35 A on-column assembled MEDS-AB-Tn5 as in (Picelli et al., 2014, supra): FIG. 35 B ) as FIG. 35 A ) but exposed to T4-PNK reaction conditions for 30 min at 37° C.;
• FIG. 35 C as FIG. 35 B ) but including T4-PNK enzyme;
• FIG. 35 D a bar chart showing the quantification of the relative proportions of nucleosome-protected fragments recovered in FIGS. 35 A-C .
• FIGS. 36 A-B shows photographs of arrays generated according to the workflow in FIG. 28 , depicting the ligation efficiency of DNA tagments onto capture probe oligonucleotides ( FIG. 36 A ) without and ( FIG. 36 B ) with post-assembly phosphorylation.
• FIG. 37 is a schematic depicting a representative embodiment of the invention in which tagments are gap-filled with a polymerase with slippery activity (e.g., stuttering), creating poly-A-sticky end (3′ overhang) at the 3′-ends (mimicking an mRNA poly(A)-tail) with a terminal transferase and subsequent hybridization to the capture domain of a capture probe (this embodiment would allow simultaneous hybridization of mRNA-transcripts).
• a polymerase can be used to extend the tagment prior to capture.
• FIG. 38 is a schematic diagram of a representative embodiment of the invention in which tagments are ligated to partially double stranded capture probes using the capture domain strand of the capture probe (e.g., a capture domain oligonucleotide) as a ligation template.
• the capture domain strand of the capture probe e.g., a capture domain oligonucleotide
• FIG. 39 is a schematic diagram showing a representative workflow of the procedure used to investigate ligation of phosphorylated DNA tagments from a whole human genome and downstream qPCR analysis.
• FIG. 40 shows a schematic representation of an exemplary oligonucleotide capture strategy and the respective sequences. Readout is performed by qPCR with oligonucleotides specific to tagments successfully ligated to the surface (e.g., A-short and Nextera reverse) or to all tagments (e.g., Nextera forward and Nextera reverse).
• tagments successfully ligated to the surface
• tagments e.g., A-short and Nextera reverse
• All tagments e.g., Nextera forward and Nextera reverse.
• FIG. 41 A is a schematic diagram of a substrate outline under various experimental conditions following the workflow shown in FIG. 39 (ligation of phosphorylated DNA fragments from a whole human genome).
• FIG. 41 B shows a DNA fragment analysis of tagmentation reactions performed according to the workflow shown in FIG. 39 .
• the PCR primer pair “Ashort-Next” covers both the surface probe and the tagment. This primer pair only results in a PCR product when hybridization and ligation have occurred.
• Samples 1 and 2 represent tagments with phosphate groups added to facilitate ligation.
• Samples 3 and 4 had tagments lacking phosphate groups and served as negative controls and samples 5 and 6 had MQ water instead of tagments.
• a pair of Nextera primers (“NEXT ONLY”, samples 7-11) show the PCR products when both ligation and hybridization have occurred, thus resulting in a signal from the D and E wells.
• FIG. 41 C shows a graph showing an alignment of PCR products.
• the graph shows ligation (ligated qPCR products) with “Ashort-Next” primers, whereas minimal ligation occurred in all four negative controls.
• FIG. 42 shows a schematic diagram showing a representative workflow of the procedure used to investigate permeabilization and tagmentation conditions of DNA tagments in immobilized tissue sections. Results from partial protein digestion with trypsin or Proteinase-K during pre-permeabilization are shown.
• FIGS. 43 A-C shows graphs showing the effect of collagenase treatment followed by either Proteinase-K ( FIG. 43 A ) or trypsin ( FIG. 43 B ) pre-permeabilization on tagmentation efficiency according to the workflow shown in FIG. 42 .
• the experiment was performed in duplicate. Proteinase-K pre-permeabilization treatment resulted in uniformly high signal of amplified tagments compared to trypsin pre-permeabilization treatment or ( FIG. 43 C ) the negative control (phosphate negative tagments).
• FIG. 44 shows a schematic diagram showing a representative workflow of the procedure used to investigate the capture of DNA tagments from immobilized tissue sections.
• FIGS. 45 A-D shows graphs and photographs showing the successful capture of DNA tagments from immobilized tissue sections according to the workflow shown in FIG. 44 with collagenase and Proteinase-K pre-permeabilization treatment.
• Each experiment was performed in duplicate: one experiment for PCR downstream analysis and one experiment for hybridization using a fluorescently labeled (Cy5) oligonucleotide complementary to the ligated tagments.
• the phosphate positive samples resulted in detectable signal ( FIG. 45 A and FIG. 45 B ), whereas the phosphate negative sample did not ( FIG. 45 C ).
• FIG. 45 D shows a hematoxylin-eosin image (left) and the corresponding spatial pattern of ligated DNA tagments (right) showing successful DNA capture from the tissue section.
• FIG. 46 A is a schematic diagram showing an example sample handling apparatus that can be used to implement various steps and methods described herein.
• FIG. 46 B is a schematic diagram showing an example imaging apparatus that can be used to obtain images of biological samples, analytes, and arrays of features.
• FIG. 46 C is a schematic diagram of an example of a control unit of the apparatus of FIGS. 46 A and 46 B .
• This disclosure describes apparatus, systems, methods, and compositions for spatial analysis of biological samples. This section in particular describes certain general terminology, analytes, sample types, and preparative steps that are referred to in later sections of the disclosure.
• Tissues and cells can be obtained from any source.
• tissues and cells can be obtained from single-cell or multicellular organisms (e.g., a mammal).
• Tissues and cells obtained from a mammal e.g., a human, often have varied analyte levels (e.g., gene and/or protein expression) which can result in differences in cell morphology and/or function.
• the position of a cell within a tissue can affect, e.g., the cell's fate, behavior, morphology, and signaling and cross-talk with other cells in the tissue.
• Information regarding the differences in analyte levels (gene and/or protein expression) within different cells in a tissue of a mammal can also help physicians select or administer a treatment that will be effective in the single-cell or multicellular organisms (e.g., a mammal) based on the detected differences in analyte levels within different cells in the tissue.
• Differences in analyte levels within different cells in a tissue of a mammal can also provide information on how tissues (e.g., healthy and diseased tissues) function and/or develop.
• Differences in analyte levels within different cells in a tissue of a mammal can also provide information of different mechanisms of disease pathogenesis in a tissue and mechanism of action of a therapeutic treatment within a tissue.
• Differences in analyte levels within different cells in a tissue of a mammal can also provide information on drug resistance mechanisms and the development of the same in a tissue of a mammal. Differences in the presence or absence of analytes within different cells in a tissue of a multicellular organism (e.g., a mammal) can provide information on drug resistance mechanisms and the development of the same in a tissue of a multicellular organism.
• the spatial analysis methodologies provide for the detection of differences in an analyte level (e.g., gene and/or protein expression) within different cells in a tissue of a mammal or within a single cell from a mammal.
• spatial analysis methodologies can be used to detect the differences in analyte levels (e.g., gene and/or protein expression) within different cells in histological slide samples, the data from which can be reassembled to generate a three-dimensional map of analyte levels (e.g., gene and/or protein expression) of a tissue sample obtained from a mammal, e.g., with a degree of spatial resolution (e.g., single-cell resolution).
• a degree of spatial resolution e.g., single-cell resolution
• RNA-seq RNA-seq
• Such approaches however, rely on a relatively small set of pre-defined markers, therefore introducing selection bias that limits discovery.
• RNA-seq RNA-seq
• Spatial RNA assays traditionally relied on staining for a limited number of RNA species.
• single-cell RNA-sequencing allows for deep profiling of cellular gene expression (including non-coding RNA), but the established methods separate cells from their native spatial context.
• Spatial analysis methods include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the position of the capture probe within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte (e.g., a protein and/or nucleic acid) produced by and/or present in a cell.
• a spatial barcode e.g., a nucleic acid sequence that provides information as to the position of the capture probe within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample)
• an analyte e.g., a protein and/or nucleic acid
• the spatial barcode can be a nucleic acid that has a unique sequence, a unique fluorophore or a unique combination of fluorophores, a unique amino acid sequence, a unique heavy metal or a unique combination of heavy metals, or any other unique detectable agent.
• the capture domain can be any agent that is capable of binding to an analyte produced by and/or present in a cell (e.g., a nucleic acid that is capable of hybridizing to a nucleic acid from a cell (e.g., an mRNA, genomic DNA, mitochondrial DNA, or miRNA), a substrate or binding partner of an analyte, or an antibody that binds specifically to an analyte).
• a capture probe can also include a nucleic acid sequence that is complementary to a sequence of a universal forward and/or universal reverse primer.
• a capture probe can also include a cleavage site (e.g., a cleavage recognition site of a restriction endonuclease), a photolabile bond, a thermosensitive bond, or a chemical-sensitive bond.
• the binding of an analyte to a capture probe can be detected using a number of different methods, e.g., nucleic acid sequencing, fluorophore detection, nucleic acid amplification, detection of nucleic acid ligation, and/or detection of nucleic acid cleavage products.
• the detection is used to associate a specific spatial barcode with a specific analyte produced by and/or present in a cell (e.g., a mammalian cell).
• Capture probes can be, e.g., attached to a surface, e.g., a solid array, a bead, or a coverslip. In some examples, capture probes are not attached to a surface. In some examples, capture probes can be encapsulated within, embedded within, or layered on a surface of a permeable composition (e.g., any of the substrates described herein). For example, capture probes can be encapsulated or disposed within a permeable bead (e.g., a gel bead). In some examples, capture probes can be encapsulated within, embedded within, or layered on a surface of a substrate (e.g., any of the exemplary substrates described herein, such as a hydrogel or a porous membrane).
• a permeable composition e.g., any of the substrates described herein.
• capture probes can be encapsulated or disposed within a permeable bead (e.g., a
• a cell or a tissue sample including a cell are contacted with capture probes attached to a substrate (e.g., a surface of a substrate), and the cell or tissue sample is permeabilized to allow analytes to be released from the cell and bind to the capture probes attached to the substrate.
• analytes released from a cell can be actively directed to the capture probes attached to a substrate using a variety of methods, e.g., electrophoresis, chemical gradient, pressure gradient, fluid flow, or magnetic field.
• a capture probe can be directed to interact with a cell or a tissue sample using a variety of methods, e.g., inclusion of a lipid anchoring agent in the capture probe, inclusion of an agent that binds specifically to, or forms a covalent bond with a membrane protein in the capture probe, fluid flow, pressure gradient, chemical gradient, or magnetic field.
• Non-limiting aspects of spatial analysis methodologies are described in WO 2011/127099, WO 2014/210233, WO 2014/210225, WO 2016/162309, WO 2018/091676, WO 2012/140224, WO 2014/060483, U.S. Pat. Nos. 10,002,316, 9,727,810, U.S. Patent Application Publication No. 2017/0016053, Rodriques et al., Science 363(6434):1463-1467, 2019; WO 2018/045186, Lee et al., Nat. Protoc.
• a “barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and/or a capture probe).
• a barcode can be part of an analyte, or independent of an analyte.
• a barcode can be attached to an analyte.
• a particular barcode can be unique relative to other barcodes.
• Barcodes can have a variety of different formats.
• barcodes can include polynucleotide barcodes, random nucleic acid and/or amino acid sequences, and synthetic nucleic acid and/or amino acid sequences.
• a barcode can be attached to an analyte or to another moiety or structure in a reversible or irreversible manner.
• a barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample.
• Barcodes can allow for identification and/or quantification of individual sequencing-reads (e.g., a barcode can be or can include a unique molecular identifier or “UMI”).
• Barcodes can spatially-resolve molecular components found in biological samples, for example, at single-cell resolution (e.g., a barcode can be or can include a “spatial barcode”).
• a barcode includes both a UMI and a spatial barcode.
• a barcode includes two or more sub-barcodes that together function as a single barcode.
• a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) that are separated by one or more non-barcode sequences.
• nucleic acid and “nucleotide” are intended to be consistent with their use in the art and to include naturally-occurring species or functional analogs thereof. Particularly useful functional analogs of nucleic acids are capable of hybridizing to a nucleic acid in a sequence-specific fashion (e.g., capable of hybridizing to two nucleic acids such that ligation can occur between the two hybridized nucleic acids) or are capable of being used as a template for replication of a particular nucleotide sequence.
• Naturally-occurring nucleic acids generally have a backbone containing phosphodiester bonds.
• An analog structure can have an alternate backbone linkage including any of a variety of those known in the art.
• Naturally-occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g. found in ribonucleic acid (RNA)).
• a deoxyribose sugar e.g., found in deoxyribonucleic acid (DNA)
• RNA ribonucleic acid
• a nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are known in the art.
• a nucleic acid can include native or non-native nucleotides.
• a native deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C), or guanine (G)
• a ribonucleic acid can have one or more bases selected from the group consisting of uracil (U), adenine (A), cytosine (C), or guanine (G).
• Useful non-native bases that can be included in a nucleic acid or nucleotide are known in the art.
• a “probe” or a “target,” when used in reference to a nucleic acid or sequence of a nucleic acids, is intended as a semantic identifier for the nucleic acid or sequence in the context of a method or composition, and does not limit the structure or function of the nucleic acid or sequence beyond what is expressly indicated.
• oligonucleotide and “polynucleotide” are used interchangeably to refer to a single-stranded multimer of nucleotides from about 2 to about 500 nucleotides in length. Oligonucleotides can be synthetic, made enzymatically (e.g., via polymerization), or using a “split-pool” method. Oligonucleotides can include ribonucleotide monomers (i.e., can be oligoribonucleotides) and/or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides).
• oligonucleotides can include a combination of both deoxyribonucleotide monomers and ribonucleotide monomers in the oligonucleotide (e.g., random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers).
• An oligonucleotide can be 4 to 10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, or 400-500 nucleotides in length, for example.
• Oligonucleotides can include one or more functional moieties that are attached (e.g., covalently or non-covalently) to the multimer structure.
• an oligonucleotide can include one or more detectable labels (e.g., a radioisotope or fluorophore).
• a “subject” is an animal, such as a mammal (e.g., human or a non-human simian), or avian (e.g., bird), or other organism, such as a plant.
• a mammal such as a rodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate (i.e.
• a plant such as Arabidopsis thaliana , corn, sorghum, oat, wheat, rice, canola, or soybean; an algae such as Chlamydomonas reinhardtii ; a nematode such as Caenorhabditis elegans ; an insect such as Drosophila melanogaster , mosquito, fruit fly, or honey bee; an arachnid such as a spider; a fish such as zebrafish; a reptile; an amphibian such as a frog or Xenopus laevis ; a Dictyostelium discoideum; a fungi such as Pneumocystis carinii, Takifugu rubripes , yeast, Saccharamoyces cerevisiae or Schizosaccharomyces pombe ; or a Plasmodium falciparum.
• a plant such as Arabidopsis thaliana , corn,
• a “genome” generally refers to genomic information from a subject, which can be, for example, at least a portion of, or the entirety of, the subject's gene-encoded hereditary information.
• a genome can include coding regions (e.g., that code for proteins) as well as non-coding regions.
• a genome can include the sequences of some or all of the subject's chromosomes. For example, the human genome ordinarily has a total of 46 chromosomes. The sequences of some or all of these can constitute the genome.
• an “adaptor,” an “adapter,” and a “tag” are terms that are used interchangeably in this disclosure, and refer to species that can be coupled to a polynucleotide sequence (in a process referred to as “tagging”) using any one of many different techniques including (but not limited to) ligation, hybridization, and tagmentation.
• Adaptors can also be nucleic acid sequences that add a function, e.g., spacer sequences, primer sequences/sites, barcode sequences, unique molecular identifier sequences.
• hybridizing refers to the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. Pairing can be achieved by any process in which a nucleic acid sequence joins with a substantially or fully complementary sequence through base pairing to form a hybridization complex.
• two nucleic acid sequences are “substantially complementary” if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of their individual bases are complementary to one another.
• a “primer” is a single-stranded nucleic acid sequence having a 3′ end that can be used as a substrate for a nucleic acid polymerase in a nucleic acid extension reaction.
• RNA primers are formed of RNA nucleotides, and are used in RNA synthesis, while DNA primers are formed of DNA nucleotides and used in DNA synthesis.
• Primers can also include both RNA nucleotides and DNA nucleotides (e.g., in a random or designed pattern). Primers can also include other natural or synthetic nucleotides described herein that can have additional functionality.
• DNA primers can be used to prime RNA synthesis and vice versa (e.g., RNA primers can be used to prime DNA synthesis).
• Primers can vary in length. For example, primers can be about 6 bases to about 120 bases. For example, primers can include up to about 25 bases.
• a “primer extension” refers to any method where two nucleic acid sequences (e.g., a constant region from each of two distinct capture probes) become linked (e.g., hybridized) by an overlap of their respective terminal complementary nucleic acid sequences (i.e., for example, 3′ termini). Such linking can be followed by nucleic acid extension (e.g., an enzymatic extension) of one, or both termini using the other nucleic acid sequence as a template for extension. Enzymatic extension can be performed by an enzyme including, but not limited to, a polymerase and/or a reverse transcriptase.
• a “proximity ligation” is a method of ligating two (or more) nucleic acid sequences that are in proximity with each other through enzymatic means (e.g., a ligase).
• proximity ligation can include a “gap-filling” step that involves incorporation of one or more nucleic acids by a polymerase, based on the nucleic acid sequence of a template nucleic acid molecule, spanning a distance between the two nucleic acid molecules of interest (see, e.g., U.S. Pat. No. 7,264,929, the entire contents of which are incorporated herein by reference).
• a wide variety of different methods can be used for proximity ligating nucleic acid molecules, including (but not limited to) “sticky-end” and “blunt-end” ligations.
• single-stranded ligation can be used to perform proximity ligation on a single-stranded nucleic acid molecule.
• Sticky-end proximity ligations involve the hybridization of complementary single-stranded sequences between the two nucleic acid molecules to be joined, prior to the ligation event itself.
• Blunt-end proximity ligations generally do not include hybridization of complementary regions from each nucleic acid molecule because both nucleic acid molecules lack a single-stranded overhang at the site of ligation.
• a “nucleic acid extension” generally involves incorporation of one or more nucleic acids (e.g., A, G, C, T, U, nucleotide analogs, or derivatives thereof) into a molecule (such as, but not limited to, a nucleic acid sequence) in a template-dependent manner, such that consecutive nucleic acids are incorporated by an enzyme (such as a polymerase or reverse transcriptase), thereby generating a newly synthesized nucleic acid molecule.
• an enzyme such as a polymerase or reverse transcriptase
• a primer that hybridizes to a complementary nucleic acid sequence can be used to synthesize a new nucleic acid molecule by using the complementary nucleic acid sequence as a template for nucleic acid synthesis.
• a 3′ polyadenylated tail of an mRNA transcript that hybridizes to a poly (dT) sequence can be used as a template for single-strand synthesis of a corresponding cDNA molecule.
• PCR amplification refers to the use of a polymerase chain reaction (PCR) to generate copies of genetic material, including DNA and RNA sequences. Suitable reagents and conditions for implementing PCR are described, for example, in U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,512,462, the entire contents of each of which are incorporated herein by reference.
• the reaction mixture includes the genetic material to be amplified, an enzyme, one or more primers that are employed in a primer extension reaction, and reagents for the reaction.
• the oligonucleotide primers are of sufficient length to provide for hybridization to complementary genetic material under annealing conditions.
• the length of the primers generally depends on the length of the amplification domains, but will typically be at least 4 bases, at least 5 bases, at least 6 bases, at least 8 bases, at least 9 bases, at least 10 base pairs (bp), at least 11 bp, at least 12 bp, at least 13 bp, at least 14 bp, at least 15 bp, at least 16 bp, at least 17 bp, at least 18 bp, at least 19 bp, at least 20 bp, at least 25 bp, at least 30 bp, at least 35 bp, and can be as long as 40 bp or longer, where the length of the primers will generally range from 18 to 50 bp.
• the genetic material can be contacted with a single primer or a set of two primers (forward and reverse primers), depending upon whether primer extension, linear or exponential amplification of the genetic material is desired.
• the PCR amplification process uses a DNA polymerase enzyme.
• the DNA polymerase activity can be provided by one or more distinct DNA polymerase enzymes.
• the DNA polymerase enzyme is from a bacterium, e.g., the DNA polymerase enzyme is a bacterial DNA polymerase enzyme.
• the DNA polymerase can be from a bacterium of the genus Escherichia, Bacillus, Thermophilus , or Pyrococcus.
• DNA polymerases that can be used include, but are not limited to: E. coli DNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, VENTTM DNA polymerase ( Thermococcus litoralis -derived polymerase), DEEPVENTTM DNA polymerase ( Pyrococcus sp. GB-D-derived polymerase), LongAmp® Taq DNA polymerase (blend of Thermus aquaticus YT-1-derived polymerase and Pyrococcus sp.
• GB-D-derived polymerase LongAmp® Hot Start Taq DNA polymerase (blend of aptamer-based Thermus aquaticus YT-1-derived polymerase and Pyrococcus sp. GB-D-derived polymerase), Crimson LongAmp® Taq DNA polymerase (blend of Thermus aquaticus YT-1-derived polymerase, Pyrococcus sp. GB-D-derived polymerase, and a colored reaction buffer), Crimson Taq DNA polymerase ( Thermus aquaticus YT-1-derived polymerase and a colored reaction buffer), OneTaq® DNA polymerase (blend of Thermus aquaticus YT-1-derived polymerase and Pyrococcus sp.
• GB-D-derived polymerase OneTaq® QuickLoad® DNA polymerase (blend of Thermus aquaticus YT-1-derived polymerase, Pyrococcus sp. GB-D-derived polymerase, and two loading dyes), Hemo KlenTaq® DNA polymerase (truncated variant of Thermus aquaticus -derived polymerase well-suited for whole blood samples), REDTaq® DNA polymerase ( Thermus aquaticus -derived polymerase and a loading dye), Phusion® DNA polymerase (a Pyrococcus -like enzyme fused with a processivity-enhancing DNA-binding domain), Phusion® High-Fidelity DNA polymerase (a Pyrococcus -like enzyme fused with a processivity-enhancing DNA-binding domain), Platinum Pfx DNA polymerase ( Thermococcus sp.
• KOD-derived polymerase KOD-derived polymerase
• AccuPrime Pfx DNA polymerase Thermococcus sp. KOD-derived polymerase
• Phi29 DNA polymerase Phi29 DNA polymerase
• Klenow fragment Pwo DNA polymerase
• Pfu DNA polymerase Pfu DNA polymerase
• T4 DNA polymerase T7 DNA polymerase enzymes.
• DNA polymerase includes not only naturally-occurring enzymes but also all modified derivatives thereof, including also derivatives of naturally-occurring DNA polymerase enzymes.
• the DNA polymerase can have been modified to remove 5′-3′ exonuclease activity.
• Sequence-modified derivatives or mutants of DNA polymerase enzymes that can be used include, but are not limited to, mutants that retain at least some of the functional, e.g. DNA polymerase activity of the wild-type sequence. Mutations can affect the activity profile of the enzymes, e.g. enhance or reduce the rate of polymerization, under different reaction conditions, e.g. temperature, template concentration, primer concentration, etc. Mutations or sequence-modifications can also affect the exonuclease activity and/or thermostability of the enzyme.
• PCR amplification can include reactions such as, but not limited to, a strand-displacement amplification reaction, a rolling circle amplification reaction, a ligase chain reaction, a transcription-mediated amplification reaction, an isothermal amplification reaction, and/or a loop-mediated amplification reaction.
• reactions such as, but not limited to, a strand-displacement amplification reaction, a rolling circle amplification reaction, a ligase chain reaction, a transcription-mediated amplification reaction, an isothermal amplification reaction, and/or a loop-mediated amplification reaction.
• PCR amplification uses a single primer that is complementary to the 3′ tag of target DNA fragments. In some embodiments, PCR amplification uses a first and a second primer, where at least a 3′ end portion of the first primer is complementary to at least a portion of the 3′ tag of the target nucleic acid fragments, and where at least a 3′ end portion of the second primer exhibits the sequence of at least a portion of the 5′ tag of the target nucleic acid fragments.
• a 5′ end portion of the first primer is non-complementary to the 3′ tag of the target nucleic acid fragments, and a 5′ end portion of the second primer does not exhibit the sequence of at least a portion of the 5′ tag of the target nucleic acid fragments.
• the first primer includes a first universal sequence and/or the second primer includes a second universal sequence.
• the PCR amplification products can be ligated to additional sequences using a DNA ligase enzyme.
• the DNA ligase activity can be provided by one or more distinct DNA ligase enzymes.
• the DNA ligase enzyme is from a bacterium, e.g., the DNA ligase enzyme is a bacterial DNA ligase enzyme.
• the DNA ligase enzyme is from a virus (e.g., a bacteriophage).
• the DNA ligase can be T4 DNA ligase.
• Other enzymes appropriate for the ligation step include, but are not limited to, Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain 9oN) DNA ligase (9oNtm DNA ligase, available from New England Biolabs, Ipswich, Mass.), and AmpligaseTM (a thermostable DNA ligase available from Epicentre Biotechnologies, Madison, Wis.). Derivatives, e.g. sequence-modified derivatives, and/or mutants thereof, can also be used.
• genetic material is amplified by reverse transcription polymerase chain reaction (RT-PCR).
• the desired reverse transcriptase activity can be provided by one or more distinct reverse transcriptase enzymes, suitable examples of which include, but are not limited to: M-MLV, MuLV, AMV, HIV, ArrayScriptTM (a modified M-MLV reverse transcriptase), MultiScribeTM (a modified MoMuLV reverse transcriptase), ThermoScriptTM (a modified avian reverse transcriptase), and SuperScript® I, II, III, and IV enzymes (a series of modified MMLV reverse transcriptases).
• Reverse transcriptase includes not only naturally occurring enzymes, but all such modified derivatives thereof, including also derivatives of naturally-occurring reverse transcriptase enzymes.
• reverse transcription can be performed using sequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIV reverse transcriptase enzymes, including mutants that retain at least some of the functional, e.g. reverse transcriptase, activity of the wild-type sequence.
• the reverse transcriptase enzyme can be provided as part of a composition that includes other components, e.g. stabilizing components that enhance or improve the activity of the reverse transcriptase enzyme, such as RNase inhibitor(s), inhibitors of DNA-dependent DNA synthesis, e.g. actinomycin D.
• Many sequence-modified derivative or mutants of reverse transcriptase enzymes e.g.
• M-MLV and compositions including unmodified and modified enzymes are commercially available, e.g. ArrayScriptTM (a modified M-MLV reverse transcriptase), MultiScribeTM (a modified MoMuLV reverse transcriptase), ThermoScriptTM (a modified avian reverse transcriptase), and SuperScript® I, II, III, and IV enzymes (a series of modified MMLV reverse transcriptases).
• ArrayScriptTM a modified M-MLV reverse transcriptase
• MultiScribeTM a modified MoMuLV reverse transcriptase
• ThermoScriptTM a modified avian reverse transcriptase
• SuperScript® I, II, III, and IV enzymes a series of modified MMLV reverse transcriptases
• Certain reverse transcriptase enzymes can synthesize a complementary DNA strand using both RNA (cDNA synthesis) and single-stranded DNA (ssDNA) as a template.
• the reverse transcription reaction can use an enzyme (reverse transcriptase) that is capable of using both RNA and ssDNA as the template for an extension reaction, e.g. an AMV or MMLV reverse transcriptase.
• the quantification of RNA and/or DNA is carried out by real-time PCR (also known as quantitative PCR or qPCR), using techniques well known in the art, such as but not limited to “TAQMANTM” (dual labeled hydrolysis probes) or “SYBR®” (high-sensitivity dye for staining DNA and RNA), or on capillaries (“LightCycler® Capillaries”)(device used to head and cool biological samples).
• the quantification of genetic material is determined by optical absorbance and with real-time PCR.
• the quantification of genetic material is determined by digital PCR.
• the genes analyzed can be compared to a reference nucleic acid extract (DNA and RNA) corresponding to the expression (mRNA) and quantity (DNA) in order to compare expression levels of the target nucleic acids.
• an “antibody” is a polypeptide molecule that recognizes and binds to a complementary target antigen. Antibodies typically have a molecular structure shape that resembles a Y shape. Naturally-occurring antibodies, referred to as immunoglobulins, belong to one of the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE. Antibodies can also be produced synthetically. For example, recombinant antibodies, which are monoclonal antibodies, can be synthesized using synthetic genes by recovering the antibody genes from source cells, amplifying into an appropriate vector, and introducing the vector into a host to cause the host to express the recombinant antibody.
• recombinant antibodies can be cloned from any species of antibody-producing animal using suitable oligonucleotide primers and/or hybridization probes. Recombinant techniques can be used to generate antibodies and antibody fragments, including non-endogenous species.
• Synthetic antibodies can be derived from non-immunoglobulin sources.
• antibodies can be generated from nucleic acids (e.g., aptamers), and from non-immunoglobulin protein scaffolds (such as peptide aptamers) into which hypervariable loops are inserted to form antigen binding sites.
• nucleic acids e.g., aptamers
• non-immunoglobulin protein scaffolds such as peptide aptamers
• Synthetic antibodies based on nucleic acids or peptide structures can be smaller than immunoglobulin-derived antibodies, leading to greater tissue penetration.
• Antibodies can also include affimer proteins, which are affinity reagents that typically have a molecular weight of about 12-14 kDa.
• Affimer proteins generally bind to a target (e.g., a target protein) with both high affinity and specificity. Examples of such targets include, but are not limited to, ubiquitin chains, immunoglobulins, and C-reactive protein.
• affimer proteins are derived from cysteine protease inhibitors, and include peptide loops and a variable N-terminal sequence that provides the binding site.
• Antibodies can also include single domain antibodies (VHH domains and VNAR domains), scFvs, and Fab fragments.
• an “affinity group” is a molecule or molecular moiety which has a high affinity or preference for associating or binding with another specific or particular molecule or moiety.
• the association or binding with another specific or particular molecule or moiety can be via a non-covalent interaction, such as hydrogen bonding, ionic forces, and van der Waals interactions.
• An affinity group can, for example, be biotin, which has a high affinity or preference to associate or bind to the protein avidin or streptavidin.
• An affinity group for example, can also refer to avidin or streptavidin which has an affinity to biotin.
• an affinity group and specific or particular molecule or moiety to which it binds or associates with include, but are not limited to, antibodies or antibody fragments and their respective antigens, such as digoxigenin and anti-digoxigenin antibodies, lectin, and carbohydrates (e.g., a sugar, a monosaccharide, a disaccharide, or a polysaccharide), and receptors and receptor ligands.
• antibodies or antibody fragments and their respective antigens such as digoxigenin and anti-digoxigenin antibodies, lectin, and carbohydrates (e.g., a sugar, a monosaccharide, a disaccharide, or a polysaccharide), and receptors and receptor ligands.
• affinity group and its specific or particular molecule or moiety to which it binds or associates with can have their roles reversed, for example, such that between a first molecule and a second molecule, in a first instance the first molecule is characterized as an affinity group for the second molecule, and in a second instance the second molecule is characterized as an affinity group for the first molecule.
• detectable label refers to a directly or indirectly detectable moiety that is associated with (e.g., conjugated to) a molecule to be detected, e.g., a capture probe or analyte.
• the detectable label can be directly detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can be indirectly detectable, e.g., by catalyzing chemical alterations of a substrate compound or composition, which substrate compound or composition is directly detectable.
• Detectable labels can be suitable for small scale detection and/or suitable for high-throughput screening.
• suitable detectable labels include, but are not limited to, radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes.
• the detectable label can be qualitatively detected (e.g., optically or spectrally), or it can be quantified.
• Qualitative detection generally includes a detection method in which the existence or presence of the detectable label is confirmed, whereas quantifiable detection generally includes a detection method having a quantifiable (e.g., numerically reportable) value such as an intensity, duration, polarization, and/or other properties.
• the detectable label is bound to a feature or to a capture probe associated with a feature.
• detectably labeled features can include a fluorescent, a colorimetric, or a chemiluminescent label attached to a bead (see, for example, Rajeswari et al., J. Microbiol Methods 139:22-28, 2017, and Forcucci et al., J. Biomed Opt. 10:105010, 2015, the entire contents of each of which are incorporated herein by reference).
• a plurality of detectable labels can be attached to a feature, capture probe, or composition to be detected.
• detectable labels can be incorporated during 25 nucleic acid polymerization or amplification (e.g., Cy5® (tetramethylindo(di)-carbocyanine dye with excitation wavelength of about 646 nm)-labelled nucleotides, such as Cy5®dCTP). Any suitable detectable label can be used.
• the detectable label is a fluorophore.
• the fluorophore can be from a group that includes: 7-AAD (7-Aminoactinomycin D), Acridine Orange (+DNA), Acridine Orange (+RNA), Alexa Fluor® 350 (a fluorophore with an excitation wavelength of about 350 nm), Alexa Fluor® 430 (a fluorophore with an excitation wavelength of about 430 nm), Alexa Fluor® 488 (a fluorophore with an excitation wavelength of about 488 nm), Alexa Fluor® 532 (a fluorophore with an excitation wavelength of about 532 nm), Alexa Fluor® 546 (a fluorophore with an excitation wavelength of about 546 nm), Alexa Fluor® 555 (a fluorophore with an excitation wavelength of about 555 nm), Alexa Fluor® 568 (a fluorophore with an excitation wavelength of about 568 nm), Alexa Fluor® 594 (a fluorophore with an excitation wavelength of about
• a detectable label is or includes a luminescent or chemiluminescent moiety.
• luminescent/chemiluminescent moieties include, but are not limited to, peroxidases such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and luciferase. These protein moieties can catalyze chemiluminescent reactions given the appropriate substrates (e.g., an oxidizing reagent plus a chemiluminescent compound. A number of compound families are known to provide chemiluminescence under a variety of conditions.
• Non-limiting examples of chemiluminescent compound families include 2,3-dihydro-1,4-phthalazinedione luminol, 5-amino-6,7,8-trimethoxy- and the dimethylamino[ca]benz analog. These compounds can luminesce in the presence of alkaline hydrogen peroxide or calcium hypochlorite and base.
• chemiluminescent compound families include, e.g., 2,4,5-triphenylimidazoles, para-dimethylamino and -methoxy substituents, oxalates such as oxalyl active esters, p-nitrophenyl, N-alkyl acridinum esters, luciferins, lucigenins, or acridinium esters.
• a “template switching oligonucleotide” is an oligonucleotide that hybridizes to untemplated nucleotides added by a reverse transcriptase (e.g., enzyme with terminal transferase activity) during reverse transcription.
• a template switching oligonucleotide hybridizes to untemplated poly(C) nucleotides added by a reverse transcriptase.
• the template switching oligonucleotide adds a common 5′ sequence to full-length cDNA that is used for cDNA amplification.
• the template switching oligonucleotide adds a common sequence onto the 5′ end of the RNA being reverse transcribed.
• a template switching oligonucleotide can hybridize to untemplated poly(C) nucleotides added onto the end of a cDNA molecule and provide a template for the reverse transcriptase to continue replication to the 5′ end of the template switching oligonucleotide, thereby generating full-length cDNA ready for further amplification.
• the template switching oligonucleotide can serve as a primer in a cDNA amplification reaction.
• a template switching oligonucleotide is added before, contemporaneously with, or after a reverse transcription, or other terminal transferase-based reaction. In some embodiments, a template switching oligonucleotide is included in the capture probe. In certain embodiments, methods of sample analysis using template switching oligonucleotides can involve the generation of nucleic acid products from analytes of the tissue sample, followed by further processing of the nucleic acid products with the template switching oligonucleotide.
• Template switching oligonucleotides can include a hybridization region and a template region.
• the hybridization region can include any sequence capable of hybridizing to the target.
• the hybridization region can, e.g., include a series of G bases to complement the overhanging C bases at the 3′ end of a cDNA molecule.
• the series of G bases can include 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases, or more than 5 G bases.
• the template sequence can include any sequence to be incorporated into the cDNA.
• the hybridization region can include at least one base in addition to at least one G base.
• the hybridization can include bases that are not a G base.
• the template region includes at least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequences and/or functional sequences.
• the template region and hybridization region are separated by a spacer.
• the template regions include a barcode sequence.
• the barcode sequence can act as a spatial barcode and/or as a unique molecular identifier.
• Template switching oligonucleotides can include deoxyribonucleic acids; ribonucleic acids; modified nucleic acids including 2-aminopurine, 2,6-diaminopurine (2-amino-dA), inverted dT, 5-methyl dC, 2′-deoxyInosine, Super T (5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), or any combination of the fore
• the length of a template switching oligonucleotide can be at least about 1, 2, 10, 20, 50, 75, 100, 150, 200, or 250 nucleotides or longer. In some embodiments, the length of a template switching oligonucleotide can be at most about 2, 10, 20, 50, 100, 150, 200, or 250 nucleotides or longer.
• a “splint oligonucleotide” is an oligonucleotide that, when hybridized to other polynucleotides, acts as a “splint” to position the polynucleotides next to one another so that they can be ligated together.
• the splint oligonucleotide is DNA or RNA.
• the splint oligonucleotide can include a nucleotide sequence that is partially complimentary to nucleotide sequences from two or more different oligonucleotides.
• the splint oligonucleotide assists in ligating a “donor” oligonucleotide and an “acceptor” oligonucleotide.
• an RNA ligase, a DNA ligase, or another other variety of ligase is used to ligate two nucleotide sequences together
• the splint oligonucleotide is between 10 and 50 oligonucleotides in length, e.g., between 10 and 45, 10 and 40, 10 and 35, 10 and 30, 10 and 25, or 10 and 20 oligonucleotides in length.
• the splint oligonucleotide is between 15 and 50, 15 and 45, 15 and 40, 15 and 35, 15 and 30, 15 and 30, or 15 and 25 nucleotides in length.
• an “analyte” can include any biological substance, structure, moiety, or component to be analyzed.
• target can similarly refer to an analyte of interest.
• Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes.
• non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral coat proteins, extracellular and intracellular proteins, antibodies, and antigen binding fragments.
• the analyte can be an organelle (e.g., nuclei or mitochondria).
• Cell surface features corresponding to analytes can include, but are not limited to, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, an extracellular matrix protein, a posttranslational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation or lipidation) state of a cell surface protein, a gap junction, and an adherens junction.
• a posttranslational modification e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, ace
• Analytes can be derived from a specific type of cell and/or a specific sub-cellular region.
• analytes can be derived from cytosol, from cell nuclei, from mitochondria, from microsomes, and more generally, from any other compartment, organelle, or portion of a cell.
• Permeabilizing agents that specifically target certain cell compartments and organelles can be used to selectively release analytes from cells for analysis.
• nucleic acid analytes examples include DNA analytes such as genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA/DNA hybrids.
• nucleic acid analytes also include RNA analytes such as various types of coding and non-coding RNA.
• RNA analytes such as various types of coding and non-coding RNA.
• examples of the different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA.
• the RNA can be a transcript (e.g., present in a tissue section).
• the RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length).
• Small RNAs mainly include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA).
• the RNA can be double-stranded RNA or single-stranded RNA.
• the RNA can be circular RNA.
• the RNA can be a bacterial rRNA (e.g., 16s rRNA or 23s rRNA).
• analytes include mRNA and cell surface features (e.g., using the labelling agents described herein), mRNA and intracellular proteins (e.g., transcription factors), mRNA and cell methylation status, mRNA and accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq), mRNA and metabolites (e.g., using the labelling agents described herein), a barcoded labelling agent (e.g., the oligonucleotide tagged antibodies described herein) and a V(D)J sequence of an immune cell receptor (e.g., T-cell receptor), mRNA and a perturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisense oligonucleotide as described herein).
• mRNA and cell surface features e.g., using the labelling agents described herein
• Analytes can include a nucleic acid molecule with a nucleic acid sequence encoding at least a portion of a V(D)J sequence of an immune cell receptor (e.g., a TCR or BCR).
• the nucleic acid molecule is cDNA first generated from reverse transcription of the corresponding mRNA, using a poly(T) containing primer. The generated cDNA can then be barcoded using a capture probe, featuring a barcode sequence (and optionally, a UMI sequence) that hybridizes with at least a portion of the generated cDNA.
• a template switching oligonucleotide hybridizes to a poly(C) tail added to a 3′end of the cDNA by a reverse transcriptase enzyme.
• the original mRNA template and template switching oligonucleotide can then be denatured from the cDNA and the barcoded capture probe can then hybridize with the cDNA and a complement of the cDNA generated.
• Additional methods and compositions suitable for barcoding cDNA generated from mRNA transcripts including those encoding V(D)J regions of an immune cell receptor and/or barcoding methods and composition including a template switch oligonucleotide are described in PCT Patent Application PCT/US2017/057269, filed Oct. 18, 2017, and U.S. patent application Ser.
• V(D)J analysis can also be completed with the use of one or more labelling agents that bind to particular surface features of immune cells and associated with barcode sequences.
• the one or more labelling agents can include an MHC or MHC multimer.
• the analyte can include a nucleic acid capable of functioning as a component of a gene editing reaction, such as, for example, clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing.
• the capture probe can include a nucleic acid sequence that is complementary to the analyte (e.g., a sequence that can hybridize to the CRISPR RNA (crRNA), single guide RNA (sgRNA), or an adapter sequence engineered into a crRNA or sgRNA).
• an analyte can be extracted from a live cell. Processing conditions can be adjusted to ensure that a biological sample remains live during analysis, and analytes are extracted from (or released from) live cells of the sample. Live cell-derived analytes can be obtained only once from the sample, or can be obtained at intervals from a sample that continues to remain in viable condition.
• the systems, apparatus, methods, and compositions can be used to analyze any number of analytes.
• the number of analytes that are analyzed can be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000 or more different analytes present in a region of the sample or within an individual feature of the substrate.
• Methods for performing multiplexed assays to analyze two or more different analytes will be discussed in a subsequent section of this disclosure.
• a “biological sample” is obtained from the subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and/or other biological material from the subject.
• a biological sample can also be obtained from a prokaryote such as a bacterium, e.g., Escherichia coli , Staphylococci or Mycoplasma pneumoniae ; an archaea; a virus such as Hepatitis C virus or human immunodeficiency virus; or a viroid.
• a biological sample can be obtained from non-mammalian organisms (e.g., a plants, an insect, an arachnid, a nematode, a fungi, or an amphibian).
• a biological sample can also be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX).
• PDO patient derived organoid
• PDX patient derived xenograft
• Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., a patient with a disease such as cancer) or a pre-disposition to a disease, and/or individuals that are in need of therapy or suspected of needing therapy.
• the biological sample can include any number of macromolecules, for example, cellular macromolecules and organelles (e.g., mitochondria and nuclei).
• the biological sample can be a nucleic acid sample and/or protein sample.
• the biological sample can be a carbohydrate sample or a lipid sample.
• the biological sample can be obtained as a tissue sample, such as a tissue section, biopsy, a core biopsy, needle aspirate, or fine needle aspirate.
• the sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample.
• the sample can be a skin sample, a colon sample, a cheek swab, a histology sample, a histopathology sample, a plasma or serum sample, a tumor sample, living cells, cultured cells, a clinical sample such as, for example, whole blood or blood-derived products, blood cells, or cultured tissues or cells, including cell suspensions.
• Cell-free biological samples can include extracellular polynucleotides.
• Extracellular polynucleotides can be isolated from a bodily sample, e.g., blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears.
• Bio samples can be derived from a homogeneous culture or population of the subjects or organisms mentioned herein or alternatively from a collection of several different organisms, for example, in a community or ecosystem.
• Biological samples can include one or more diseased cells.
• a diseased cell can have altered metabolic properties, gene expression, protein expression, and/or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells.
• Biological samples can also include fetal cells.
• a procedure such as amniocentesis can be performed to obtain a fetal cell sample from maternal circulation.
• Sequencing of fetal cells can be used to identify any of a number of genetic disorders, including, e.g., aneuploidy such as Down's syndrome, Edwards syndrome, and Patau syndrome.
• cell surface features of fetal cells can be used to identify any of a number of disorders or diseases.
• Biological samples can also include immune cells. Sequence analysis of the immune repertoire of such cells, including genomic, proteomic, and cell surface features, can provide a wealth of information to facilitate an understanding the status and function of the immune system. By way of example, determining the status (e.g., negative or positive) of minimal residue disease (MRD) in a multiple myeloma (MM) patient following autologous stem cell transplantation is considered a predictor of MRD in the MM patient (see, e.g., U.S. Patent Application Publication No. 2018/0156784, the entire contents of which are incorporated herein by reference).
• MRD minimal residue disease
• immune cells in a biological sample include, but are not limited to, B cells, T cells (e.g., cytotoxic T cells, natural killer T cells, regulatory T cells, and T helper cells), natural killer cells, cytokine induced killer (CTK) cells, myeloid cells, such as granulocytes (basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes/hypersegmented neutrophils), monocytes/macrophages, mast cells, thrombocytes/megakaryocytes, and dendritic cells.
• T cells e.g., cytotoxic T cells, natural killer T cells, regulatory T cells, and T helper cells
• CTK cytokine induced killer
• myeloid cells such as granulocytes (basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes/hypersegmented neutrophils), monocytes/macrophages, mast cells, throm
• a biological sample can include a single analyte of interest, or more than one analyte of interest.
• Methods for performing multiplexed assays to analyze two or more different analytes in a single biological sample will be discussed in a subsequent section of this disclosure.
• a biological sample can be harvested from a subject (e.g., via surgical biopsy, whole subject sectioning) or grown in vitro on a growth substrate or culture dish as a population of cells, and prepared for analysis as a tissue slice or tissue section. Grown samples may be sufficiently thin for analysis without further processing steps. Alternatively, grown samples, and samples obtained via biopsy or sectioning, can be prepared as thin tissue sections using a mechanical cutting apparatus such as a vibrating blade microtome. As another alternative, in some embodiments, a thin tissue section can be prepared by applying a touch imprint of a biological sample to a suitable substrate material.
• the thickness of the tissue section can be a fraction of (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) the maximum cross-sectional dimension of a cell.
• tissue sections having a thickness that is larger than the maximum cross-section cell dimension can also be used.
• cryostat sections can be used, which can be, e.g., 10-20 micrometers thick.
• the thickness of a tissue section typically depends on the method used to prepare the section and the physical characteristics of the tissue, and therefore sections having a wide variety of different thicknesses can be prepared and used.
• the thickness of the tissue section can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40, or 50 micrometers.
• Thicker sections can also be used if desired or convenient, e.g., at least 70, 80, 90, or 100 micrometers or more.
• the thickness of a tissue section is between 1-100 micrometers, 1-50 micrometers, 1-30 micrometers, 1-25 micrometers, 1-20 micrometers, 1-15 micrometers, 1-10 micrometers, 2-8 micrometers, 3-7 micrometers, or 4-6 micrometers, but as mentioned above, sections with thicknesses larger or smaller than these ranges can also be analysed.
• Multiple sections can also be obtained from a single biological sample.
• multiple tissue sections can be obtained from a surgical biopsy sample by performing serial sectioning of the biopsy sample using a sectioning blade. Spatial information among the serial sections can be preserved in this manner, and the sections can be analysed successively to obtain three-dimensional information about the biological sample.
• the biological sample e.g., a tissue section as described above
• a temperature suitable to maintain or preserve the integrity e.g., the physical characteristics
• a temperature can be, e.g., less than ⁇ 20° C., or less than ⁇ 25° C., ⁇ 30° C., ⁇ 40° C., ⁇ 50° C., ⁇ 60° C., ⁇ 70° C., ⁇ 80° C.
• the frozen tissue sample can be sectioned, e.g., thinly sliced, onto a substrate surface using any number of suitable methods.
• a tissue sample can be prepared using a chilled microtome (e.g., a cryostat) set at a temperature suitable to maintain both the structural integrity of the tissue sample and the chemical properties of the nucleic acids in the sample.
• a temperature can be, e.g., less than ⁇ 15° C., less than ⁇ 20° C., or less than ⁇ 25° C.
• the biological sample can be prepared using formalin-fixation and paraffin-embedding (FFPE), which are established methods.
• FFPE formalin-fixation and paraffin-embedding
• cell suspensions and other non-tissue samples can be prepared using formalin-fixation and paraffin-embedding.
• the sample can be sectioned as described above.
• the paraffin-embedding material can be removed from the tissue section (e.g., deparaffinization) by incubating the tissue section in an appropriate solvent (e.g., xylene) followed by a rinse (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes).
• a biological sample can be fixed in any of a variety of other fixatives to preserve the biological structure of the sample prior to analysis.
• a sample can be fixed via immersion in ethanol, methanol, acetone, paraformaldehyde-Triton, and combinations thereof.
• acetone fixation is used with fresh frozen samples, which can include, but are not limited to, cortex tissue, mouse olfactory bulb, human brain tumor, human post-mortem brain, and breast cancer samples.
• pre-permeabilization steps may not be performed.
• acetone fixation can be performed in conjunction with permeabilization steps.
• a biological sample can be embedded in any of a variety of other embedding materials to provide structural substrate to the sample prior to sectioning and other handling steps.
• the embedding material is removed prior to analysis of tissue sections obtained from the sample.
• suitable embedding materials include, but are not limited to, waxes, resins (e.g., methacrylate resins), epoxies, and agar.
• biological samples can be stained using a wide variety of stains and staining techniques.
• a sample can be stained using any number of stains, including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, haematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranine.
• stains including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, haematoxylin, Hoechst stains, iodine, methyl green, m
• the sample can be stained using hematoxylin and eosin (H&E) staining techniques, using Papanicolaou staining techniques, Masson's trichrome staining techniques, silver staining techniques, Sudan staining techniques, and/or using Periodic Acid Schiff (PAS) staining techniques.
• HPA staining is typically performed after formalin or acetone fixation.
• the sample can be stained using Romanowsky stain, including Wright's stain, Jenner's stain, Can-Grunwald stain, Leishman stain, and Giemsa stain.
• biological samples can be destained.
• Methods of destaining or discoloring a biological sample are known in the art, and generally depend on the nature of the stain(s) applied to the sample.
• one or more immunofluorescent stains are applied to the sample via antibody coupling.
• Such stains can be removed using techniques such as cleavage of disulfide linkages via treatment with a reducing agent and detergent washing, chaotropic salt treatment, treatment with antigen retrieval solution, and treatment with an acidic glycine buffer.
• Methods for multiplexed staining and destaining are described, for example, in Bolognesi et al., J. Histochem. Cytochem.
• the biological sample can be embedded in a hydrogel matrix. Embedding the sample in this manner typically involves contacting the biological sample with a hydrogel such that the biological sample becomes surrounded by the hydrogel.
• the sample can be embedded by contacting the sample with a suitable polymer material, and activating the polymer material to form a hydrogel.
• the hydrogel is formed such that the hydrogel is internalized within the biological sample.
• the biological sample is immobilized in the hydrogel via cross-linking of the polymer material that forms the hydrogel.
• Cross-linking can be performed chemically and/or photochemically, or alternatively by any other hydrogel-formation method known in the art.
• the composition and application of the hydrogel-matrix to a biological sample typically depends on the nature and preparation of the biological sample (e.g., sectioned, non-sectioned, type of fixation).
• the hydrogel-matrix can include a monomer solution and an ammonium persulfate (APS) initiator/tetramethylethylenediamine (TEMED) accelerator solution.
• APS ammonium persulfate
• TEMED tetramethylethylenediamine
• the biological sample consists of cells (e.g., cultured cells or cells disassociated from a tissue sample)
• the cells can be incubated with the monomer solution and APS/TEMED solutions.
• hydrogel-matrix gels are formed in compartments, including but not limited to devices used to culture, maintain, or transport the cells.
• hydrogel-matrices can be formed with monomer solution plus APS/TEMED added to the compartment to a depth ranging from about 0.1 ⁇ m to about 2 mm.
• hydrogel embedding of biological samples are described for example in Chen et al., Science 347(6221):543-548, 2015, the entire contents of which are incorporated herein by reference.
• a biological sample embedded in a hydrogel can be isometrically expanded.
• Isometric expansion methods that can be used include hydration, a preparative step in expansion microscopy, as described in Chen et al., Science 347(6221):543-548, 2015.
• Isometric expansion can be performed by anchoring one or more components of a biological sample to a gel, followed by gel formation, proteolysis, and swelling. Isometric expansion of the biological sample can occur prior to immobilization of the biological sample on a substrate, or after the biological sample is immobilized to a substrate. In some embodiments, the isometrically expanded biological sample can be removed from the substrate prior to contacting the substrate with capture probes, as will be discussed in greater detail in a subsequent section.
• the steps used to perform isometric expansion of the biological sample can depend on the characteristics of the sample (e.g., thickness of tissue section, fixation, cross-linking), and/or the analyte of interest (e.g., different conditions to anchor RNA, DNA, and protein to a gel).
• characteristics of the sample e.g., thickness of tissue section, fixation, cross-linking
• analyte of interest e.g., different conditions to anchor RNA, DNA, and protein to a gel.
• proteins in the biological sample are anchored to a swellable gel such as a polyelectrolyte gel.
• An antibody can be directed to the protein before, after, or in conjunction with being anchored to the swellable gel.
• DNA and/or RNA in a biological sample can also be anchored to the swellable gel via a suitable linker.
• linkers include, but are not limited to, 6-((Acryloyl)amino) hexanoic acid (Acryloyl-X SE) (available from ThermoFisher, Waltham, Mass.), Label-IT Amine (available from MirusBio, Madison, Wis.) and Label X (described for example in Chen et al., Nat. Methods 13:679-684, 2016, the entire contents of which are incorporated herein by reference).
• Isometric expansion of the sample can increase the spatial resolution of the subsequent analysis of the sample.
• the increased resolution in spatial profiling can be determined by comparison of an isometrically expanded sample with a sample that has not been isometrically expanded.
• a biological sample is isometrically expanded to a size at least 2 ⁇ , 2.1 ⁇ , 2.2 ⁇ , 2.3 ⁇ , 2.4 ⁇ , 2.5 ⁇ , 2.6 ⁇ , 2.7 ⁇ , 2.8 ⁇ , 2.9 ⁇ , 3 ⁇ , 3.1 ⁇ , 3.2 ⁇ , 3.3 ⁇ , 3.4 ⁇ , 3.5 ⁇ , 3.6 ⁇ , 3.7 ⁇ , 3.8 ⁇ , 3.9 ⁇ , 4 ⁇ , 4.1 ⁇ , 4.2 ⁇ , 4.3 ⁇ , 4.4 ⁇ , 4.5 ⁇ , 4.6 ⁇ , 4.7 ⁇ , 4.8 ⁇ , or 4.9 ⁇ its non-expanded size.
• the sample is isometrically expanded to at least 2 ⁇ and less than 20 ⁇ of its non-expanded size.
• the biological sample can be attached to a substrate.
• substrates suitable for this purpose are described in detail below. Attachment of the biological sample can be irreversible or reversible, depending upon the nature of the sample and subsequent steps in the analytical method.
• the sample can be attached to the substrate reversibly by applying a suitable polymer coating to the substrate, and contacting the sample to the polymer coating.
• the sample can then be detached from the substrate using an organic solvent that at least partially dissolves the polymer coating.
• Hydrogels are examples of polymers that are suitable for this purpose.
• the substrate can be coated or functionalized with one or more substances to facilitate attachment of the sample to the substrate.
• Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, poly-lysine, antibodies, and polysaccharides.
• the biological sample corresponds to cells (e.g., derived from a cell culture or a tissue sample).
• cells e.g., derived from a cell culture or a tissue sample.
• individual cells can be naturally unaggregated.
• the cells can be derived from a suspension of cells and/or disassociated or disaggregated cells from a tissue or tissue section.
• the cells in the sample may be aggregated, and may be disaggregated into individual cells using, for example, enzymatic or mechanical techniques.
• enzymes used in enzymatic disaggregation include, but are not limited to, dispase, collagenase, trypsin, and combinations thereof.
• Mechanical disaggregation can be performed, for example, using a tissue homogenizer.
• the biological sample can be derived from a cell culture grown in vitro.
• Samples derived from a cell culture can include one or more suspension cells which are anchorage-independent within the cell culture. Examples of such cells include, but are not limited to, cell lines derived from hematopoietic cells, and from the following cell lines: Colo205, CCRF-CEM, HL-60, K562, MOLT-4, RPMI-8226, SR, HOP-92, NCI-H322M, and MALME-3M.
• Samples derived from a cell culture can include one or more adherent cells which grow on the surface of the vessel that contains the culture medium.
• adherent cells include DU145 (prostate cancer) cells, H295R (adrenocortical cancer) cells, HeLa (cervical cancer) cells, KBM-7 (chronic myelogenous leukemia) cells, LNCaP (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-468 (breast cancer) cells, PC3 (prostate cancer) cells, SaOS-2 (bone cancer) cells, SH-SY5Y (neuroblastoma, cloned from a myeloma) cells, T-47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, National Cancer Institute's 60 cancer cell line panel (NCI60), vero (African green monkey Chlorocebus kidney epithelial cell
• adherent cells are shown in Table 1 and catalogued, for example, in “A Catalog of in Vitro Cell Lines, Transplantable Animal and Human Tumors and Yeast,” The Division of Cancer Treatment and Diagnosis (DCTD), National Cancer Institute (2013), and in Abaan et al., “The exomes of the NCI-60 panel: a genomic resource for cancer biology and systems pharmacology,” Cancer Research 73(14):4372-82, 2013, the entire contents of each of which are incorporated by reference herein.
• the adherent cells are cells that correspond to one or more of the following cell lines: BT549, HS 578T, MCF7, MDA-MB-231, MDA-MB-468, T-47D, SF268, SF295, SF539, SNB-19, SNB-75, U251, Colo205, HCC 2998, HCT-116, HCT-15, HT29, KM12, SW620, 786-0, A498, ACHN, CAKI, RXF 393, SN12C, TK-10, UO-31, A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H460, NCI-H522, LOX IMVI, M14, MALME-3M, MDA-MB-435, SK-, EL-2, SK-MEL-28, SK-MEL-5, UACC-257, UACC-62, IGROVI, OVCAR-3, O
• a biological sample can be permeabilized to facilitate transfer of analytes out of the sample, and/or to facilitate transfer of species (such as capture probes) into the sample. If a sample is not permeabilized sufficiently, the amount of analyte captured from the sample may be too low to enable adequate analysis. Conversely, if the tissue sample is too permeable, the relative spatial relationship of the analytes within the tissue sample can be lost. Hence, a balance between permeabilizing the tissue sample enough to obtain good signal intensity while still maintaining the spatial resolution of the analyte distribution in the sample is desirable.
• a biological sample can be permeabilized by exposing the sample to one or more permeabilizing agents.
• Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X-100TM (nonionic detergent) or Tween-20TM (polysorbate 20)), and enzymes (e.g., trypsin, proteases).
• the biological sample can be incubated with a cellular permeabilizing agent to facilitate permeabilization of the sample.
• sample permeabilization Additional methods for sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol. 588:63-66, 2010, the entire contents of which are incorporated herein by reference. Any suitable method for sample permeabilization can generally be used in connection with the samples described herein.
• the diffusion-resistant medium can include at least one permeabilization reagent.
• the diffusion-resistant medium can include wells (e.g., micro-, nano-, or picowells) containing a permeabilization buffer or reagents.
• the diffusion-resistant medium is a hydrogel
• the hydrogel can include a permeabilization buffer.
• the hydrogel is soaked in permeabilization buffer prior to contacting the hydrogel with a sample.
• the hydrogel or other diffusion-resistant medium can contain dried reagents or monomers to deliver permeabilization reagents when the diffusion-resistant medium is applied to a biological sample.
• the diffusion-resistant medium i.e. hydrogel
• the hydrogel can be modified to both contain capture probes and deliver permeabilization reagents.
• a hydrogel film can be modified to include spatially-barcoded capture probes. The spatially-barcoded hydrogel film is then soaked in permeabilization buffer before contacting the spatially-barcoded hydrogel film to the sample.
• the spatially-barcoded hydrogel film thus delivers permeabilization reagents to a sample surface in contact with the spatially-barcoded hydrogel, enhancing analyte migration and capture.
• the spatially-barcoded hydrogel is applied to a sample and placed in a permeabilization bulk solution.
• the hydrogel film soaked in permeabilization reagents is sandwiched between a sample and a spatially-barcoded array.
• target analytes are able to diffuse through the permeabilizing reagent soaked hydrogel and hybridize or bind the capture probes on the other side of the hydrogel.
• the thickness of the hydrogel is proportional to the resolution loss.
• wells can contain spatially-barcoded capture probes and permeabilization reagents and/or buffer.
• spatially-barcoded capture probes and permeabilization reagents are held between spacers.
• the sample is punch, cut, or transferred into the well, wherein a target analyte diffuses through the permeabilization reagent/buffer and to the spatially-barcoded capture probes.
• resolution loss may be proportional to gap thickness (e.g. the amount of permeabilization buffer between the sample and the capture probes).
• the diffusion-resistant medium e.g. hydrogel
• the diffusion-resistant medium is between approximately 50-500 micrometers thick including 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 micrometers thick, or any thickness within 50 and 500 micrometers.
• permeabilization solution can be delivered to a sample through a porous membrane.
• a porous membrane is used to limit diffusive analyte losses, while allowing permeabilization reagents to reach a sample.
• Membrane chemistry and pore size can be manipulated to minimize analyte loss.
• the porous membrane may be made of glass, silicon, paper, hydrogel, polymer monoliths, or other material.
• the material may be naturally porous.
• the material may have pores or wells etched into solid material.
• the permeabilization reagents are flowed through a microfluidic chamber or channel over the porous membrane.
• the flow controls the sample's access to the permeabilization reagents.
• a porous membrane is sandwiched between a spatially-barcoded array and the sample, wherein permeabilization solution is applied over the porous membrane.
• the permeabilization reagents diffuse through the pores of the membrane and into the tissue.
• the biological sample can be permeabilized by adding one or more lysis reagents to the sample.
• suitable lysis agents include, but are not limited to, bioactive reagents such as lysis enzymes that are used for lysis of different cell types, e.g., gram positive or negative bacteria, plants, yeast, mammalian, such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and a variety of other commercially available lysis enzymes.
• lysis agents can additionally or alternatively be added to the biological sample to facilitate permeabilization.
• surfactant-based lysis solutions can be used to lyse sample cells. Lysis solutions can include ionic surfactants such as, for example, sarcosyl and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents can include, without limitation, organic solvents, chelating agents, detergents, surfactants, and chaotropic agents.
• the biological sample can be permeabilized by non-chemical permeabilization methods.
• Non-chemical permeabilization methods are known in the art.
• non-chemical permeabilization methods that can be used include, but are not limited to, physical lysis techniques such as electroporation, mechanical permeabilization methods (e.g., bead beating using a homogenizer and grinding balls to mechanically disrupt sample tissue structures), acoustic permeabilization (e.g., sonication), and thermal lysis techniques such as heating to induce thermal permeabilization of the sample.
• RNA analyte species of interest can be selectively enriched.
• one or more species of RNA of interest can be selected by addition of one or more oligonucleotides to the sample.
• the additional oligonucleotide is a sequence used for priming a reaction by a polymerase.
• one or more primer sequences with sequence complementarity to one or more RNAs of interest can be used to amplify the one or more RNAs of interest, thereby selectively enriching these RNAs.
• an oligonucleotide with sequence complementarity to the complementary strand of captured RNA can bind to the cDNA.
• biotinylated oligonucleotides with sequence complementary to one or more cDNA of interest binds to the cDNA and can be selected using biotinylation-strepavidin affinity using any of a variety of methods known to the field (e.g., streptavidin beads).
• RNA can be down-selected (e.g., removed) using any of a variety of methods.
• probes can be administered to a sample that selectively hybridize to ribosomal RNA (rRNA), thereby reducing the pool and concentration of rRNA in the sample. Subsequent application of the capture probes to the sample can result in improved capture of other types of RNA due to the reduction in non-specific RNA present in the sample.
• rRNA ribosomal RNA
• duplex-specific nuclease (DSN) treatment can remove rRNA (see, e.g., Archer, et al, Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage, BMC Genomics, 15 401, (2014), the entire contents of which are incorporated herein by reference).
• hydroxyapatite chromatography can remove abundant species (e.g., rRNA) (see, e.g., Vandernoot, V. A., cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications, Biotechniques, 53(6) 373-80, (2012), the entire contents of which are incorporated herein by reference).
• Additional reagents can be added to a biological sample to perform various functions prior to analysis of the sample.
• DNase and RNase inactivating agents or inhibitors such as proteinase K, and/or chelating agents such as EDTA, can be added to the sample.
• the sample can be treated with one or more enzymes.
• one or more endonucleases to fragment DNA DNA polymerase enzymes, and dNTPs used to amplify nucleic acids can be added.
• Other enzymes that can also be added to the sample include, but are not limited to, polymerase, transposase, ligase, and DNAse, and RNAse.
• reverse transcriptase enzymes can be added to the sample, including enzymes with terminal transferase activity, primers, and switch oligonucleotides.
• Template switching can be used to increase the length of a cDNA, e.g., by appending a predefined nucleic acid sequence to the cDNA.
• analytes in a biological sample can be pre-processed prior to interaction with a capture probe.
• polymerization reactions catalyzed by a polymerase e.g., DNA polymerase or reverse transcriptase
• a primer for the polymerization reaction includes a functional group that enhances hybridization with the capture probe.
• the capture probes can include appropriate capture domains to capture biological analytes of interest (e.g., poly(dT) sequence to capture poly(A) mRNA).
• biological analytes are pre-processed for library generation via next generation sequencing.
• analytes can be pre-processed by addition of a modification (e.g., ligation of sequences that allow interaction with capture probes).
• analytes e.g., DNA or RNA
• fragmentation techniques e.g., using transposases and/or fragmentation buffers.
• Fragmentation can be followed by a modification of the analyte.
• a modification can be the addition through ligation of an adapter sequence that allows hybridization with the capture probe.
• poly(A) tailing is performed. Addition of a poly(A) tail to RNA that does not contain a poly(A) tail can facilitate hybridization with a capture probe that includes a capture domain with a functional amount of poly(dT) sequence.
• ligation reactions catalyzed by a ligase are performed in the biological sample.
• ligation can be performed by chemical ligation.
• the ligation can be performed using click chemistry as further below.
• the capture domain includes a DNA sequence that has complementarity to a RNA molecule, where the RNA molecule has complementarity to a second DNA sequence, and where the RNA-DNA sequence complementarity is used to ligate the second DNA sequence to the DNA sequence in the capture domain. In these embodiments, direct detection of RNA molecules is possible.
• target-specific reactions are performed in the biological sample.
• target specific reactions include, but are not limited to, ligation of target specific adaptors, probes and/or other oligonucleotides, target specific amplification using primers specific to one or more analytes, and target-specific detection using in situ hybridization, DNA microscopy, and/or antibody detection.
• a capture probe includes capture domains targeted to target-specific products (e.g., amplification or ligation).
• This section of the disclosure describes methods, apparatus, systems, and compositions for spatial array-based analysis of biological samples.
• Array-based spatial analysis methods involve the transfer of one or more analytes from a biological sample to an array of features on a substrate, each of which is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of each analyte within the sample. The spatial location of each analyte within the sample is determined based on the feature to which each analyte is bound in the array, and the feature's relative spatial location within the array.
• FIG. 1 depicts an exemplary embodiment of this general method.
• the spatially-barcoded array populated with capture probes (as described further herein) is contacted with a sample 101 , and sample is permeabilized, allowing the target analyte to migrate away from the sample and toward the array.
• the target analyte interacts with a capture probe on the spatially-barcoded array 102 .
• the sample is optionally removed from the array and the capture probes are analyzed in order to obtain spatially-resolved analyte information 103 .
• FIG. 2 depicts an exemplary embodiment of this general method, the spatially-barcoded array populated with capture probes (as described further herein) can be contacted with a sample 201 .
• the spatially-barcoded capture probes are cleaved and then interact with cells within the provided sample 202 .
• the interaction can be a covalent or non-covalent cell-surface interaction.
• the interaction can be an intracellular interaction facilitated by a delivery system or a cell penetration peptide.
• the sample can be optionally removed for analysis.
• the sample can be optionally dissociated before analysis.
• the capture probes can be analyzed to obtain spatially-resolved information about the tagged cell 203 .
• FIG. 3 shows an exemplary workflow that includes preparing a sample on a spatially-barcoded array 301 .
• Sample preparation may include placing the sample on a slide, fixing the sample, and/or staining the sample for imaging.
• the stained sample is then imaged on the array 302 using both brightfield (to image the sample hematoxylin and eosin stain) and fluorescence (to image features) modalities.
• target analytes are then released from the sample and capture probes forming the spatially-barcoded array hybridize or bind the released target analytes 303 .
• the sample is then removed from the array 304 and the capture probes cleaved from the array 305 .
• the sample and array are then optionally imaged a second time in both modalities 305 B while the analytes are reverse transcribed into cDNA, and an amplicon library is prepared 306 and sequenced 307 .
• the two sets of images are then spatially-overlaid in order to correlate spatially-identified sample information 308 .
• a spot coordinate file is supplied by the manufacturer instead.
• the spot coordinate file replaces the second imaging step 305 B.
• amplicon library preparation 306 can be performed with a unique PCR adapter and sequenced 307 .
• FIG. 4 shows another exemplary workflow that utilizes a spatially-labelled array on a substrate, where capture probes labelled with spatial barcodes are clustered at areas called features.
• the spatially-labelled capture probes can include a cleavage domain, one or more functional sequences, a spatial barcode, a unique molecular identifier, and a capture domain.
• the spatially-labelled capture probes can also include a 5′ end modification for reversible attachment to the substrate.
• the spatially-barcoded array is contacted with a sample 401 , and the sample is permeabilized through application of permeabilization reagents 402 . Permeabilization reagents may be administered by placing the array/sample assembly within a bulk solution.
• permeabilization reagents may be administered to the sample via a diffusion-resistant medium and/or a physical barrier such as a lid, wherein the sample is sandwiched between the diffusion-resistant medium and/or barrier and the array-containing substrate.
• the analytes are migrated toward the spatially-barcoded capture array using any number of techniques disclosed herein.
• analyte migration can occur using a diffusion-resistant medium lid and passive migration.
• analyte migration can be active migration, using an electrophoretic transfer system, for example.
• the capture probes can hybridize or otherwise bind a target analyte 403 .
• the sample can be optionally removed from the array 404 .
• the capture probes can be optionally cleaved from the array 405 , and the captured analytes can be spatially-tagged by performing a reverse transcriptase first strand cDNA reaction.
• a first strand cDNA reaction can be optionally performed using template switching oligonucleotides.
• a template switching oligonucleotide can hybridize to a poly(C) tail added to a 3′end of the cDNA by a reverse transcriptase enzyme.
• the original mRNA template and template switching oligonucleotide can then be denatured from the cDNA and the barcoded capture probe can then hybridize with the cDNA and a complement of the cDNA can be generated.
• the first strand cDNA can then be purified and collected for downstream amplification steps.
• the first strand cDNA can be amplified using PCR 406 , wherein the forward and reverse primers flank the spatial barcode and target analyte regions of interest, generating a library associated with a particular spatial barcode.
• the cDNA comprises a sequencing by synthesis (SBS) primer sequence.
• SBS sequencing by synthesis
• FIG. 5 depicts an exemplary workflow where the sample is removed from the spatially-barcoded array and the spatially-barcoded capture probes are removed from the array for barcoded analyte amplification and library preparation.
• Another embodiment includes performing first strand synthesis using template switching oligonucleotides on the spatially-barcoded array without cleaving the capture probes.
• sample preparation 501 and permeabilization 502 are performed as described elsewhere herein. Once the capture probes capture the target analyte(s), first strand cDNA created by template switching and reverse transcriptase 503 is then denatured and the second strand is then extended 504 .
• the second strand cDNA is then denatured from the first strand cDNA, neutralized, and transferred to a tube 505 .
• cDNA quantification and amplification can be performed using standard techniques discussed herein.
• the cDNA can then be subjected to library preparation 506 and indexing 507 , including fragmentation, end-repair, and a-tailing, and indexing PCR steps.
• the sample can be immersed in 100% chilled methanol and incubated for 30 minutes at ⁇ 20° C. After 20 minutes, the sample can be removed and rinsed in ultrapure water. After rinsing the sample, fresh eosin solution is prepared, and the sample can be covered in isopropanol. After incubating the sample in isopropanol for 1 minute, the reagent can be removed by holding the slide at an angle, where the bottom edge of the slide can be in contact with a laboratory wipe and air dried. The sample can be uniformly covered in hematoxylin solution and incubated for 7 minutes at room temperature.
• the reagent can be removed by holding the slide at an angle, where the bottom edge of the slide can be in contact with a laboratory wipe.
• the slide containing the sample can be immersed in water and the excess liquid can be removed.
• the sample can be covered with blueing buffer and can be incubated for 2 minutes at room temperature.
• the slide containing the sample can again be immersed in water, and uniformly covered with eosin solution and incubated for 1 minute at room temperature.
• the slide can be air-dried and incubated for 5 minutes at 37° C.
• the sample can be imaged using the methods disclosed herein.
• the sample can be exposed to a permeabilization enzyme and incubated for 6 minutes at 37° C. Other permeabilization methods are described herein.
• the permeabilization enzyme can be removed and the sample prepared for analyte capture by adding SSC buffer.
• the sample can then subjected to a pre-equilibration thermocycling protocol and the SSC buffer can be removed.
• a Master Mix containing nuclease-free water, a reverse transcriptase reagent, a template switch oligo, a reducing agent, and a reverse transcriptase enzyme can be added, and the sample with the Master Mix can be subjected to a thermocycling protocol.
• the reagents can be removed from the sample and NaOH can be applied and incubated for 5 minutes at room temperature.
• the NaOH can be removed and elution buffer can be added and removed from the sample.
• a Second Strand Mix including a second strand reagent, a second strand primer, and a second strand enzyme, can be added to the sample and the sample can be sealed and incubated.
• the reagents can be removed and elution buffer can be added and removed from the sample, and NaOH can be added again to the sample and the sample can be incubated for 10 minutes at room temperature.
• Tris-HCl can be added and the reagents can be mixed.
• a qPCR Mix including nuclease-free water, qPCR Master Mix, and cDNA primers, can be prepared and the NaOH/Tris-HCl mix can be mixed with the qPCR Mix and the sample, and thermocycled according to a predetermined thermocycling protocol.
• a cDNA amplification mix can be prepared and combined with the sample and mixed. The sample can then be incubated and thermocycled. The sample can then be resuspended in SPRIselect Reagent and pipetted to ensure proper mixing.
• the sample can then be incubated at 5 minutes at room temperature, and cleared by placing the sample on a magnet (e.g., the magnet is in the high position).
• the supernatant can be removed and 80% ethanol can be added to the pellet, and incubated for 30 seconds.
• the ethanol can be removed and the pellet can be washed again.
• the sample can then be centrifuged and placed on a magnet (e.g., the magnet is on the low position). Any remaining ethanol can be removed and the sample can be air dried.
• the magnet can be removed and elution buffer can be added to the sample, mixed, and incubated for 2 minutes at room temperature.
• the sample can then be placed on the magnet (e.g., on high position) until the solution clears.
• a portion of the sample can be run on an Agilent Bioanalyzer High Sensitivity chip, where a region can be selected and the cDNA concentration can be measured to calculate the total cDNA yield.
• the quantification can be determined by Agilent Bioanalyzer or Agilent TapeStation.
• a Fragmentation Mix including a fragmentation buffer and fragmentation enzyme, can be prepared on ice. Elution buffer and fragmentation mix can be added to each sample, mixed, and centrifuged. The sample mix can then be placed in a thermocycler and cycled according to a predetermined protocol. The SPRIselect Reagent can be added to the sample and incubated at 5 minutes at room temperature. The sample can be placed on a magnet (e.g., in the high position) until the solution clears, and the supernatant can be transferred to a new tube strip. SPRIselect Reagent can be added to the sample, mixed, and incubated for 5 minutes at room temperature.
• the sample can be placed on a magnet (e.g., in the high position) until the solution clears.
• the supernatant can be removed and 80% ethanol can be added to the pellet, the pellet can be incubated for 30 seconds, and the ethanol can be removed.
• the ethanol wash can be repeated and the sample placed on a magnet (e.g., in the low position) until the solution clears.
• the remaining ethanol can be removed and elution buffer can be added to the sample, mixed, and incubated for 2 minutes at room temperature.
• the sample can be placed on a magnet (e.g., in the high position) until the solution clears, and a portion of the sample can be moved to a new tube strip.
• An Adaptor Ligation Mix including ligation buffer, DNA ligase, and adaptor oligos, can be prepared and centrifuged.
• the Adaptor Ligation Mix can be added to the sample, pipette-mixed, and centrifuged briefly.
• the sample can then be thermocycled according to a predetermined protocol.
• the SPRIsleect Reagent can be added to the sample, incubated for 5 minutes at room temperature, and placed on a magnet (e.g., in the high position) until the solution clears.
• the supernatant can be removed and the pellet can be washed with 80% ethanol, incubated for 30 seconds, and the ethanol can be removed.
• the ethanol wash can be repeated, and the sample can be centrifuged briefly before placing the sample on a magnet (e.g., in the low position). Any remaining ethanol can be removed and the sample can be air dried.
• Elution buffer can be added to the sample, the sample can be removed from the magnet, and the sample can be pipette-mixed, incubated for 2 minutes at room temperature, and placed on a magnet (e.g., in the low position) until the solution clears. A portion of the sample can be transferred to a new tube strip.
• a Sample Index PCR Mix including amplification mix and SI primer, can be prepared and combined with the sample.
• the sample/Sample Index PCR Mix can be loaded into an individual Chromium i7 Sample Index well and a thermocycling protocol can be used.
• SPRIselect Reagent can be added to each sample, mixed, and incubated for 5 minutes at room temperature.
• the sample can be placed on a magnet (e.g., in the high position) until the solution clears, and the supernatant can be transferred to a new tube strip.
• the SPRIselect Reagent can be added to each sample, pipette-mixed, and incubated for 5 minutes at room temperature.
• the sample can then be placed on a magnet (e.g., in the high position) until the solution clears.
• the supernatant can be removed, and the pellet can be washed with 80% ethanol, incubated for 30 seconds, and then the ethanol can be removed.
• the ethanol wash can be repeated, the sample centrifuged, and placed on a magnet (e.g., in the low position) to remove any remaining ethanol.
• the sample can be removed from the magnet and Elution Buffer can be added to the sample, pipette-mixed, and incubated at 2 minutes at room temperature.
• the sample can be placed on a magnet (e.g., in the low position) until the solution clears and a portion of the sample can be transferred to a new tube strip.
• the average fragment size can be determined using a Bioanalyzer trace or an Agilent TapeStation.
• performing correlative analysis of data produced by this workflow, and other workflows described herein can yield over 95% correlation of genes expressed across two capture areas (e.g. 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater).
• correlative analysis of the data can yield over 90% (e.g. over 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) correlation of genes expressed across two capture areas.
• a “capture probe” refers to any molecule capable of capturing (directly or indirectly) and/or labelling an analyte of interest in a biological sample.
• the capture probe is a nucleic acid or a polypeptide.
• the capture probe is a conjugate (e.g., an oligonucleotide-antibody conjugate).
• the capture probe includes a barcode (e.g., a spatial barcode and/or a unique molecular identifier (UMI)) and a capture domain.
• UMI unique molecular identifier
• FIG. 6 is a schematic diagram showing an example of a capture probe, as described herein.
• the capture probe 602 is optionally coupled to a feature 601 by a cleavage domain 603 , such as a disulfide linker.
• the capture probe can include functional sequences that are useful for subsequent processing, such as functional sequence 604 , which can include a sequencer specific flow cell attachment sequence, e.g., a P5 sequence, as well as functional sequence 606 , which can include sequencing primer sequences, e.g., a R1 primer binding site.
• sequence 604 is a P7 sequence and sequence 606 is a R2 primer binding site.
• a spatial barcode 605 can be included within the capture probe for use in barcoding the target analyte.
• the functional sequences can generally be selected for compatibility with any of a variety of different sequencing systems, e.g., 454 Sequencing, Ion Torrent Proton or PGM, Illumina X10, PacBio, Nanopore, etc., and the requirements thereof.
• functional sequences can be selected for compatibility with non-commercialized sequencing systems. Examples of such sequencing systems and techniques, for which suitable functional sequences can be used, include (but are not limited to) Roche 454 sequencing, Ion Torrent Proton or PGM sequencing, Illumina X10 sequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing.
• functional sequences can be selected for compatibility with other sequencing systems, including non-commercialized sequencing systems.
• the spatial barcode 605 , functional sequences 604 (e.g., flow cell attachment sequence) and 606 (e.g., sequencing primer sequences) can be common to all of the probes attached to a given feature.
• the spatial barcode can also include a capture domain 607 to facilitate capture of a target analyte.
• each capture probe includes at least one capture domain.
• the “capture domain” is an oligonucleotide, a polypeptide, a small molecule, or any combination thereof, that binds specifically to a desired analyte.
• a capture domain can be used to capture or detect a desired analyte.
• the capture domain is a functional nucleic acid sequence configured to interact with one or more analytes, such as one or more different types of nucleic acids (e.g., RNA molecules and DNA molecules).
• the functional nucleic acid sequence can include an N-mer sequence (e.g., a random N-mer sequence), which N-mer sequences are configured to interact with a plurality of DNA molecules.
• the functional sequence can include a poly(T) sequence, which poly(T) sequences are configured to interact with messenger RNA (mRNA) molecules via the poly(A) tail of an mRNA transcript.
• the functional nucleic acid sequence is the binding target of a protein (e.g., a transcription factor, a DNA binding protein, or a RNA binding protein), where the analyte of interest is a protein.
• Capture probes can include ribonucleotides and/or deoxyribonucleotides as well as synthetic nucleotide residues that are capable of participating in Watson-Crick type or analogous base pair interactions.
• the capture domain is capable of priming a reverse transcription reaction to generate cDNA that is complementary to the captured RNA molecules.
• the capture domain of the capture probe can prime a DNA extension (polymerase) reaction to generate DNA that is complementary to the captured DNA molecules.
• the capture domain can template a ligation reaction between the captured DNA molecules and a surface probe that is directly or indirectly immobilized on the substrate.
• the capture domain can be ligated to one strand of the captured DNA molecules.
• SplintR ligase along with RNA or DNA sequences (e.g., degenerate RNA) can be used to ligate a single-stranded DNA or RNA to the capture domain.
• ligases with RNA-templated ligase activity e.g., SplintR ligase, T4 RNA ligase 2 or KOD ligase, can be used to ligate a single-stranded DNA or RNA to the capture domain.
• a capture domain includes a splint oligonucleotide.
• a capture domain captures a splint oligonucleotide.
• the capture domain is located at the 3′ end of the capture probe and includes a free 3′ end that can be extended, e.g. by template dependent polymerization, to form an extended capture probe as described herein.
• the capture domain includes a nucleotide sequence that is capable of hybridizing to nucleic acid, e.g. RNA or other analyte, present in the cells of the tissue sample contacted with the array.
• the capture domain can be selected or designed to bind selectively or specifically to a target nucleic acid.
• the capture domain can be selected or designed to capture mRNA by way of hybridization to the mRNA poly(A) tail.
• the capture domain includes a poly(T) DNA oligonucleotide, i.e., a series of consecutive deoxythymidine residues linked by phosphodiester bonds, which is capable of hybridizing to the poly(A) tail of mRNA.
• the capture domain can include nucleotides that are functionally or structurally analogous to a poly(T) tail.
• a poly(U) oligonucleotide or an oligonucleotide included of deoxythymidine analogues includes at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides.
• the capture domain includes at least 25, 30, or 35 nucleotides.
• random sequences can be used to form all or a part of the capture domain.
• random sequences can be used in conjunction with poly(T) (or poly(T) analogue) sequences.
• a capture domain includes a poly(T) (or a “poly(T)-like”) oligonucleotide
• it can also include a random oligonucleotide sequence (e.g., “poly(T)-random sequence” probe). This can, for example, be located 5′ or 3′ of the poly(T) sequence, e.g. at the 3′ end of the capture domain.
• the poly(T)-random sequence probe can facilitate the capture of the mRNA poly(A) tail.
• the capture domain can be an entirely random sequence.
• degenerate capture domains can be used.
• a pool of two or more capture probes form a mixture, where the capture domain of one or more capture probes includes a poly(T) sequence and the capture domain of one or more capture probes includes random sequences. In some embodiments, a pool of two or more capture probes form a mixture where the capture domain of one or more capture probes includes poly(T)-like sequence and the capture domain of one or more capture probes includes random sequences. In some embodiments, a pool of two or more capture probes form a mixture where the capture domain of one or more capture probes includes a poly(T)-random sequences and the capture domain of one or more capture probes includes random sequences. In some embodiments, probes with degenerate capture domains can be added to any of the preceding combinations listed herein. In some embodiments, probes with degenerate capture domains can be substituted for one of the probes in each of the pairs described herein.
• the capture domain can be based on a particular gene sequence or particular motif sequence or common/conserved sequence, that it is designed to capture (i.e., a sequence-specific capture domain).
• the capture domain is capable of binding selectively to a desired sub-type or subset of nucleic acid, for example a particular type of RNA, such as mRNA, rRNA, tRNA, SRP RNA, tmRNA, snRNA, snoRNA, SmY RNA, scaRNA, gRNA, RNase P, RNase MRP, TERC, SL RNA, aRNA, cis-NAT, crRNA, lncRNA, miRNA, piRNA, siRNA, shRNA, tasiRNA, rasiRNA, 7SK, eRNA, ncRNA or other types of RNA.
• the capture domain can be capable of binding selectively to a desired subset of ribonucleic acids, for example, microbiome
• a capture domain includes an “anchor” or “anchoring sequence”, which is a sequence of nucleotides that is designed to ensure that the capture domain hybridizes to the intended biological analyte.
• an anchor sequence includes a sequence of nucleotides, including a 1-mer, 2-mer, 3-mer or longer sequence.
• the short sequence is random.
• a capture domain including a poly(T) sequence can be designed to capture an mRNA.
• an anchoring sequence can include a random 3-mer (e.g., GGG) that helps ensure that the poly(T) capture domain hybridizes to an mRNA.
• an anchoring sequence can be VN, N, or NN.
• the sequence can be designed using a specific sequence of nucleotides.
• the anchor sequence is at the 3′ end of the capture domain. In some embodiments, the anchor sequence is at the 5′ end of the capture domain.
• capture domains of capture probes are blocked prior to contacting the biological sample with the array, and blocking probes are used when the nucleic acid in the biological sample is modified prior to its capture on the array.
• the blocking probe is used to block or modify the free 3′ end of the capture domain.
• blocking probes can be hybridized to the capture probes to mask the free 3′ end of the capture domain, e.g., hairpin probes or partially double stranded probes.
• the free 3′ end of the capture domain can be blocked by chemical modification, e.g., addition of an azidomethyl group as a chemically reversible capping moiety such that the capture probes do not include a free 3′ end.
• Blocking or modifying the capture probes, particularly at the free 3′ end of the capture domain, prior to contacting the biological sample with the array, prevents modification of the capture probes, e.g., prevents the addition of a poly(A) tail to the free 3′ end of the capture probes.
• Non-limiting examples of 3′ modifications include dideoxy C-3′ (3′-ddC), 3′ inverted dT, 3′ C3 spacer, 3′Amino, and 3′ phosphorylation.
• the nucleic acid in the biological sample can be modified such that it can be captured by the capture domain.
• an adaptor sequence including a binding domain capable of binding to the capture domain of the capture probe
• this is achieved by ligation of the adaptor sequence or extension of the nucleic acid.
• an enzyme is used to incorporate additional nucleotides at the end of the nucleic acid sequence, e.g., a poly(A) tail.
• the capture probes can be reversibly masked or modified such that the capture domain of the capture probe does not include a free 3′ end.
• the 3′ end is removed, modified, or made inaccessible so that the capture domain is not susceptible to the process used to modify the nucleic acid of the biological sample, e.g., ligation or extension.
• the capture domain of the capture probe is modified to allow the removal of any modifications of the capture probe that occur during modification of the nucleic acid molecules of the biological sample.
• the capture probes can include an additional sequence downstream of the capture domain, i.e., 3′ to the capture domain, namely a blocking domain.
• the capture domain of the capture probe can be a non-nucleic acid domain.
• suitable capture domains that are not exclusively nucleic-acid based include, but are not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the functionality of any of the capture domains described herein.
• Each capture probe can optionally include at least one cleavage domain.
• the cleavage domain represents the portion of the probe that is used to reversibly attach the probe to an array feature, as will be described further below.
• one or more segments or regions of the capture probe can optionally be released from the array feature by cleavage of the cleavage domain.
• spatial barcodes and/or universal molecular identifiers (UMIs) can be released by cleavage of the cleavage domain.
• FIG. 7 is a schematic illustrating a cleavable capture probe, wherein the cleaved capture probe can enter into a non-permeabilized cell and bind to target analytes within the sample.
• the capture probe 701 contains a cleavage domain 702 , a cell penetrating peptide 703 , a reporter molecule 704 , and a disulfide bond (—S—S—). 705 represents all other parts of a capture probe, for example a spatial barcode and a capture domain.
• the cleavage domain linking the capture probe to a feature is a disulfide bond.
• a reducing agent can be added to break the disulfide bonds, resulting in release of the capture probe from the feature.
• heating can also result in degradation of the cleavage domain and release of the attached capture probe from the array feature.
• laser radiation is used to heat and degrade cleavage domains of capture probes at specific locations.
• the cleavage domain is a photo-sensitive chemical bond (i.e., a chemical bond that dissociates when exposed to light such as ultraviolet light).
• cleavage domains include labile chemical bonds such as, but not limited to, ester linkages (e.g., cleavable with an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g., cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavable via heat), a sulfone linkage (e.g., cleavable via a base), a silyl ether linkage (e.g., cleavable via an acid), a glycosidic linkage (e.g., cleavable via an amylase), a peptide linkage (e.g., cleavable via a protease), or a phosphodiester linkage (e.g., cleavable via a nuclease (e.g., DNAase)).
• ester linkages e.g., cle
• the cleavage domain includes a sequence that is recognized by one or more enzymes capable of cleaving a nucleic acid molecule, e.g., capable of breaking the phosphodiester linkage between two or more nucleotides.
• a bond can be cleavable via other nucleic acid molecule targeting enzymes, such as restriction enzymes (e.g., restriction endonucleases).
• restriction enzymes e.g., restriction endonucleases
• the cleavage domain can include a restriction endonuclease (restriction enzyme) recognition sequence. Restriction enzymes cut double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites.
• a rare-cutting restriction enzyme i.e., enzymes with a long recognition site (at least 8 base pairs in length), is used to reduce the possibility of cleaving elsewhere in the capture probe.
• the cleavage domain includes a poly(U) sequence which can be cleaved by a mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII, commercially known as the USERTM enzyme (mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII).
• UDG Uracil DNA glycosylase
• the USERTM enzyme mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII.
• Releasable capture probes can be available for reaction once released.
• an activatable capture probe can be activated by releasing the capture probes from a feature.
• the cleavage domain includes one or more mismatch nucleotides, so that the complementary parts of the surface probe and the capture probe are not 100% complementary (for example, the number of mismatched base pairs can one, two, or three base pairs).
• mismatch is recognized, e.g., by the MutY and T7 endonuclease I enzymes, which results in cleavage of the nucleic acid molecule at the position of the mismatch.
• the cleavage domain includes a nickase recognition site or sequence.
• Nickases are endonucleases which cleave only a single strand of a DNA duplex.
• the cleavage domain can include a nickase recognition site close to the 5′ end of the surface probe (and/or the 5′ end of the capture probe) such that cleavage of the surface probe or capture probe destabilizes the duplex between the surface probe and capture probe thereby releasing the capture probe) from the feature.
• Nickase enzymes can also be used in some embodiments where the capture probe is attached to the feature directly.
• the substrate can be contacted with a nucleic acid molecule that hybridizes to the cleavage domain of the capture probe to provide or reconstitute a nickase recognition site, e.g., a cleavage helper probe.
• a nickase recognition site e.g., a cleavage helper probe.
• Such cleavage helper probes can also be used to provide or reconstitute cleavage recognition sites for other cleavage enzymes, e.g., restriction enzymes.
• nickases introduce single-stranded nicks only at particular sites on a DNA molecule, by binding to and recognizing a particular nucleotide recognition sequence.
• a number of naturally-occurring nickases have been discovered, of which at present the sequence recognition properties have been determined for at least four.
• nickases are described in U.S. Pat. No. 6,867,028, which is incorporated herein by reference in its entirety.
• any suitable nickase can be used to bind to a complementary nickase recognition site of a cleavage domain.
• the nickase enzyme can be removed from the assay or inactivated following release of the capture probes to prevent unwanted cleavage of the capture probes.
• capture domains that are not exclusively nucleic-acid based include, but are not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the functionality of any of the capture domains described herein.
• a cleavage domain is absent from the capture probe.
• substrates with attached capture probes lacking a cleavage domain are described for example in Macosko et al., (2015) Cell 161, 1202-1214, the entire contents of which are incorporated herein by reference.
• the region of the capture probe corresponding to the cleavage domain can be used for some other function.
• an additional region for nucleic acid extension or amplification can be included where the cleavage domain would normally be positioned.
• the region can supplement the functional domain or even exist as an additional functional domain.
• the cleavage domain is present but its use is optional.
• Each capture probe can optionally include at least one functional domain.
• Each functional domain typically includes a functional nucleotide sequence for a downstream analytical step in the overall analysis procedure.
• the capture probe can include a functional domain for attachment to a sequencing flow cell, such as, for example, a P5 sequence for Illumina® sequencing (next-generation sequencing system).
• the capture probe or derivative thereof can include another functional domain, such as, for example, a P7 sequence for attachment to a sequencing flow cell for Illumina® sequencing (next generation sequencing system).
• the functional domains can be selected for compatibility with a variety of different sequencing systems, e.g., 454 Sequencing, Ion Torrent Proton or PGM, Illumina X10, etc., and the requirements thereof.
• the functional domain includes a primer.
• the primer can include an R1 primer sequence for Illumina® sequencing (next-generation sequencing system), and in some embodiments, an R2 primer sequence for Illumina® sequencing (next generation sequencing system). Examples of such capture probes and uses thereof are described in U.S. Patent Publication Nos. 2014/0378345 and 2015/0376609, the entire contents of each of which are incorporated herein by reference.
• the capture probe can include one or more spatial barcodes (e.g., two or more, three or more, four or more, five or more) spatial barcodes.
• a “spatial barcode” is a contiguous nucleic acid segment or two or more non-contiguous nucleic acid segments that function as a label or identifier that conveys or is capable of conveying spatial information.
• a capture probe includes a spatial barcode that possesses a spatial aspect, where the barcode is associated with a particular location within an array or a particular location on a substrate.
• a spatial barcode can be part of an analyte, or independent from an analyte (i.e., part of the capture probe).
• a spatial barcode can be a tag attached to an analyte (e.g., a nucleic acid molecule) or a combination of a tag in addition to an endogenous characteristic of the analyte (e.g., size of the analyte or end sequence(s)).
• a spatial barcode can be unique. In some embodiments where the spatial barcode is unique, the spatial barcode functions both as a spatial barcode and as a unique molecular identifier (UMI), associated with one particular capture probe.
• UMI unique molecular identifier
• Spatial barcodes can have a variety of different formats.
• spatial barcodes can include polynucleotide spatial barcodes; random nucleic acid and/or amino acid sequences; and synthetic nucleic acid and/or amino acid sequences.
• a spatial barcode is attached to an analyte in a reversible or irreversible manner.
• a spatial barcode is added to, for example, a fragment of a DNA or RNA sample before, during, and/or after sequencing of the sample.
• a spatial barcode allows for identification and/or quantification of individual sequencing-reads.
• a spatial barcode is a used as a fluorescent barcode for which fluorescently labeled oligonucleotide probes hybridize to the spatial barcode.
• the spatial barcode is a nucleic acid sequence that does not substantially hybridize to analyte nucleic acid molecules in a biological sample. In some embodiments, the spatial barcode has less than 80% sequence identity (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity) to the nucleic acid sequences across a substantial part (e.g., 80% or more) of the nucleic acid molecules in the biological sample.
• the spatial barcode sequences can include from about 6 to about 20 or more nucleotides within the sequence of the capture probes.
• the length of a spatial barcode sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer.
• the length of a spatial barcode sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the length of a spatial barcode sequence is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.
• nucleotides can be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they can be separated into two or more separate subsequences that are separated by 1 or more nucleotides.
• Separated spatial barcode subsequences can be from about 4 to about 16 nucleotides in length. In some embodiments, the spatial barcode subsequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the spatial barcode subsequence can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the spatial barcode subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.
• the one or more spatial barcode sequences of the multiple capture probes can include sequences that are the same for all capture probes coupled to the feature, and/or sequences that are different across all capture probes coupled to the feature.
• FIG. 8 is a schematic diagram of an exemplary multiplexed spatially-labelled feature.
• the feature 801 can be coupled to spatially-barcoded capture probes, wherein the spatially-barcoded probes of a particular feature can possess the same spatial barcode, but have different capture domains designed to associate the spatial barcode of the feature with more than one target analyte.
• a feature may be coupled to four different types of spatially-barcoded capture probes, each type of spatially-barcoded capture probe possessing the spatial barcode 802 .
• One type of capture probe associated with the feature includes the spatial barcode 802 in combination with a poly(T) capture domain 803 , designed to capture mRNA target analytes.
• a second type of capture probe associated with the feature includes the spatial barcode 802 in combination with a random N-mer capture domain 804 for gDNA analysis.
• a third type of capture probe associated with the feature includes the spatial barcode 802 in combination with a capture domain complementary to the capture domain on an analyte capture agent capture agent barcode domain 805 .
• a fourth type of capture probe associated with the feature includes the spatial barcode 802 in combination with a capture probe that can specifically bind a nucleic acid molecule 806 that can function in a CRISPR assay (e.g., CRISPR/Cas9). While only four different capture probe-barcoded constructs are shown in FIG.
• capture-probe barcoded constructs can be tailored for analyses of any given analyte associated with a nucleic acid and capable of binding with such a construct.
• the schemes shown in FIG. 8 can also be used for concurrent analysis of other analytes disclosed herein, including, but not limited to: (a) mRNA, a lineage tracing construct, cell surface or intracellular proteins and metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq) cell surface or intracellular proteins and metabolites, and a perturbation agent (e.g., a CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisense oligonucleotide as described herein); (c) mRNA, cell surface or intracellular proteins and/or metabolites, a barcoded labelling agent (e.g., the MHC multimers
• Capture probes attached to a single array feature can include identical (or common) spatial barcode sequences, different spatial barcode sequences, or a combination of both. Capture probes attached to a feature can include multiple sets of capture probes. Capture probes of a given set can include identical spatial barcode sequences. The identical spatial barcode sequences can be different from spatial barcode sequences of capture probes of another set.
• the plurality of capture probes can include spatial barcode sequences (e.g., nucleic acid barcode sequences) that are associated with specific locations on a spatial array.
• a first plurality of capture probes can be associated with a first region, based on a spatial barcode sequence common to the capture probes within the first region
• a second plurality of capture probes can be associated with a second region, based on a spatial barcode sequence common to the capture probes within the second region.
• the second region may or may not be associated with the first region.
• Additional pluralities of capture probes can be associated with spatial barcode sequences common to the capture probes within other regions.
• the spatial barcode sequences can be the same across a plurality of capture probe molecules.
• multiple different spatial barcodes are incorporated into a single arrayed capture probe.
• a mixed but known set of spatial barcode sequences can provide a stronger address or attribution of the spatial barcodes to a given spot or location, by providing duplicate or independent confirmation of the identity of the location.
• the multiple spatial barcodes represent increasing specificity of the location of the particular array point.
• the capture probe can include one or more (e.g., two or more, three or more, four or more, five or more) Unique Molecular Identifiers (UMIs).
• UMIs Unique Molecular Identifiers
• a unique molecular identifier is a contiguous nucleic acid segment or two or more non-contiguous nucleic acid segments that function as a label or identifier for a particular analyte, or for a capture probe that binds a particular analyte (e.g., via the capture domain).
• a UMI can be unique.
• a UMI can include one or more specific polynucleotides sequences, one or more random nucleic acid and/or amino acid sequences, and/or one or more synthetic nucleic acid and/or amino acid sequences.
• the UMI is a nucleic acid sequence that does not substantially hybridize to analyte nucleic acid molecules in a biological sample. In some embodiments, the UMI has less than 80% sequence identity (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity) to the nucleic acid sequences across a substantial part (e.g., 80% or more) of the nucleic acid molecules in the biological sample.
• sequence identity e.g., less than 70%, 60%, 50%, or less than 40% sequence identity
• the UMI can include from about 6 to about 20 or more nucleotides within the sequence of the capture probes.
• the length of a UMI sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer.
• the length of a UMI sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer.
• the length of a UMI sequence is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.
• UMI subsequences can be from about 4 to about 16 nucleotides in length. In some embodiments, the UMI subsequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the UMI subsequence can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the UMI subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.
• a UMI is attached to an analyte in a reversible or irreversible manner.
• a UMI is added to, for example, a fragment of a DNA or RNA sample before, during, and/or after sequencing of the analyte.
• a UMI allows for identification and/or quantification of individual sequencing-reads.
• a UMI is a used as a fluorescent barcode for which fluorescently labeled oligonucleotide probes hybridize to the UMI.
• an individual array feature can include one or more capture probes.
• an individual array feature includes hundreds or thousands of capture probes.
• the capture probes are associated with a particular individual feature, where the individual feature contains a capture probe including a spatial barcode unique to a defined region or location on the array.
• a particular feature can contain capture probes including more than one spatial barcode (e.g., one capture probe at a particular feature can include a spatial barcode that is different than the spatial barcode included in another capture probe at the same particular feature, while both capture probes include a second, common spatial barcode), where each spatial barcode corresponds to a particular defined region or location on the array.
• capture probes including more than one spatial barcode e.g., one capture probe at a particular feature can include a spatial barcode that is different than the spatial barcode included in another capture probe at the same particular feature, while both capture probes include a second, common spatial barcode
• each spatial barcode corresponds to a particular defined region or location on the array.
• multiple spatial barcode sequences associated with one particular feature on an array can provide a stronger address or attribution to a given location by providing duplicate or independent confirmation of the location.
• the multiple spatial barcodes represent increasing specificity of the location of the particular array point.
• a particular array point can be coded with two different spatial barcodes, where each spatial barcode identifies a particular defined region within the array, and an array point possessing both spatial barcodes identifies the sub-region where two defined regions overlap, e.g., such as the overlapping portion of a Venn diagram.
• a particular array point can be coded with three different spatial barcodes, where the first spatial barcode identifies a first region within the array, the second spatial barcode identifies a second region, where the second region is a subregion entirely within the first region, and the third spatial barcode identifies a third region, where the third region is a subregion entirely within the first and second subregions.
• capture probes attached to array features are released from the array features for sequencing.
• capture probes remain attached to the array features, and the probes are sequenced while remaining attached to the array features (e.g., via in-situ sequencing). Further aspects of the sequencing of capture probes are described in subsequent sections of this disclosure.
• an array feature can include different types of capture probes attached to the feature.
• the array feature can include a first type of capture probe with a capture domain designed to bind to one type of analyte, and a second type of capture probe with a capture domain designed to bind to a second type of analyte.
• array features can include one or more (e.g., two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, 12 or more, 15 or more, 20 or more, 30 or more, 50 or more) different types of capture probes attached to a single array feature.
• the capture probe is nucleic acid. In some embodiments, the capture probe is attached to the array feature via its 5′ end. In some embodiments, the capture probe includes from the 5′ to 3′ end: one or more barcodes (e.g., a spatial barcode and/or a UMI) and one or more capture domains. In some embodiments, the capture probe includes from the 5′ to 3′ end: one barcode (e.g., a spatial barcode or a UMI) and one capture domain. In some embodiments, the capture probe includes from the 5′ to 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., a spatial barcode and/or a UMI), and a capture domain.
• the capture probe includes from the 5′ to 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., a spatial barcode and/or a UMI), and a capture domain.
• the capture probe includes from the 5′ to 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., a spatial barcode and/or a UMI), a second functional domain, and a capture domain.
• the capture probe includes from the 5′ to 3′ end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain.
• the capture probe does not include a spatial barcode.
• the capture probe does not include a UMI.
• the capture probe includes a sequence for initiating a sequencing reaction.
• the capture probe is immobilized on a feature via its 3′ end.
• the capture probe includes from the 3′ to 5′ end: one or more barcodes (e.g., a spatial barcode and/or a UMI) and one or more capture domains.
• the capture probe includes from the 3′ to 5′ end: one barcode (e.g., a spatial barcode or a UMI) and one capture domain.
• the capture probe includes from the 3′ to 5′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., a spatial barcode and/or a UMI), and a capture domain.
• the capture probe includes from the 3′ to 5′ end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain.
• a capture probe includes an in situ synthesized oligonucleotide.
• the in situ synthesized oligonucleotide includes one or more constant sequences, one or more of which serves as a priming sequence (e.g., a primer for amplifying target nucleic acids).
• a constant sequence is a cleavable sequence.
• the in situ synthesized oligonucleotide includes a barcode sequence, e.g., a variable barcode sequence.
• the in situ synthesized oligonucleotide is attached to a feature of an array.
• a capture probe is a product of two or more oligonucleotide sequences, e.g., two or more oligonucleotide sequences that are ligated together.
• one of the oligonucleotide sequences is an in situ synthesized oligonucleotide.
• the capture probe includes a splint oligonucleotide.
• Two or more oligonucleotides can be ligated together using a splint oligonucleotide and any variety of ligases known in the art or described herein (e.g., SplintR ligase).
• one of the oligonucleotides includes: a constant sequence (e.g., a sequence complementary to a portion of a splint oligonucleotide), a degenerate sequence, and a capture domain (e.g., as described herein).
• the capture probe is generated by having an enzyme add polynucleotides at the end of an oligonucleotide sequence.
• the capture probe can include a degenerate sequence, which can function as a unique molecular identifier.
• a capture probe can include a degenerate sequence, which is a sequence in which some positions of a nucleotide sequence contain a number of possible bases.
• a degenerate sequence can be a degenerate nucleotide sequence including about or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 nucleotides.
• a nucleotide sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 0, 10, 15, 20, 25, or more degenerate positions within the nucleotide sequence.
• the degenerate sequence is used as a UMI.
• a capture probe includes a restriction endonuclease recognition sequence or a sequence of nucleotides cleavable by specific enzyme activities.
• uracil sequences can be cleaved by specific enzyme activity.
• other modified bases e.g., modified by methylation
• the capture probes can be subjected to an enzymatic cleavage, which removes the blocking domain and any of the additional nucleotides that are added to the 3′ end of the capture probe during the modification process. The removal of the blocking domain reveals and/or restores the free 3′ end of the capture domain of the capture probe.
• additional nucleotides can be removed to reveal and/or restore the 3′ end of the capture domain of the capture probe.
• a blocking domain can be incorporated into the capture probe when it is synthesized, or after its synthesis.
• the terminal nucleotide of the capture domain is a reversible terminator nucleotide (e.g., 3′-O-blocked reversible terminator and 3′-unblocked reversible terminator), and can be included in the capture probe during or after probe synthesis.
• An “extended capture probe” is a capture probe with an enlarged nucleic acid sequence.
• an “extended 3′ end” indicates that further nucleotides were added to the most 3′ nucleotide of the capture probe to extend the length of the capture probe, for example, by standard polymerization reactions utilized to extend nucleic acid molecules including templated polymerization catalyzed by a polymerase (e.g., a DNA polymerase or reverse transcriptase).
• a polymerase e.g., a DNA polymerase or reverse transcriptase
• extending the capture probe includes generating cDNA from the captured (hybridized) RNA. This process involves synthesis of a complementary strand of the hybridized nucleic acid, e.g., generating cDNA based on the captured RNA template (the RNA hybridized to the capture domain of the capture probe).
• the captured (hybridized) nucleic acid e.g., RNA
• the capture probe is extended using reverse transcription.
• reverse transcription includes synthesizing cDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), using a reverse transcriptase.
• reverse transcription is performed while the tissue is still in place, generating an analyte library, where the analyte library includes the spatial barcodes from the adjacent capture probes.
• the capture probe is extended using one or more DNA polymerases.
• the capture domain of the capture probe includes a primer for producing the complementary strand of the nucleic acid hybridized to the capture probe, e.g., a primer for DNA polymerase and/or reverse transcription.
• the nucleic acid, e.g., DNA and/or cDNA, molecules generated by the extension reaction incorporate the sequence of the capture probe.
• the extension of the capture probe e.g., a DNA polymerase and/or reverse transcription reaction, can be performed using a variety of suitable enzymes and protocols.
• a full-length DNA, e.g. cDNA, molecule is generated.
• a “full-length” DNA molecule refers to the whole of the captured nucleic acid molecule. However, if the nucleic acid, e.g. RNA, was partially degraded in the tissue sample, then the captured nucleic acid molecules will not be the same length as the initial RNA in the tissue sample.
• the 3′ end of the extended probes, e.g., first strand cDNA molecules is modified. For example, a linker or adaptor can be ligated to the 3′ end of the extended probes.
• template switching oligonucleotides are used to extend cDNA in order to generate a full-length cDNA (or as close to a full-length cDNA as possible).
• a second strand synthesis helper probe (a partially double stranded DNA molecule capable of hybridizing to the 3′ end of the extended capture probe), can be ligated to the 3′ end of the extended probe, e.g., first strand cDNA, molecule using a double stranded ligation enzyme such as T4 DNA ligase.
• a double stranded ligation enzyme such as T4 DNA ligase.
• Other enzymes appropriate for the ligation step are known in the art and include, e.g., Tth DNA ligase, Taq DNA ligase, Thermococcus sp.
• a polynucleotide tail e.g., a poly(A) tail, is incorporated at the 3′ end of the extended probe molecules. In some embodiments, the polynucleotide tail is incorporated using a terminal transferase active enzyme.
• double-stranded extended capture probes are treated to remove any unextended capture probes prior to amplification and/or analysis, e.g. sequence analysis. This can be achieved by a variety of methods, e.g., using an enzyme to degrade the unextended probes, such as an exonuclease enzyme, or purification columns.
• extended capture probes are amplified to yield quantities that are sufficient for analysis, e.g., via DNA sequencing.
• the first strand of the extended capture probes e.g., DNA and/or cDNA molecules
• acts as a template for the amplification reaction e.g., a polymerase chain reaction.
• the amplification reaction incorporates an affinity group onto the extended capture probe (e.g., RNA-cDNA hybrid) using a primer including the affinity group.
• the primer includes an affinity group and the extended capture probes includes the affinity group.
• the affinity group can correspond to any of the affinity groups described previously.
• the extended capture probes including the affinity group can be coupled to an array feature specific for the affinity group.
• the substrate can include an antibody or antibody fragment.
• the array feature includes avidin or streptavidin and the affinity group includes biotin.
• the array feature includes maltose and the affinity group includes maltose-binding protein.
• the array feature includes maltose-binding protein and the affinity group includes maltose.
• amplifying the extended capture probes can function to release the extended probes from the array feature, insofar as copies of the extended probes are not attached to the array feature.
• the extended capture probe or complement or amplicon thereof is released from an array feature.
• the step of releasing the extended capture probe or complement or amplicon thereof from an array feature can be achieved in a number of ways.
• an extended capture probe or a complement thereof is released from the feature by nucleic acid cleavage and/or by denaturation (e.g. by heating to denature a double-stranded molecule).
• the extended capture probe or complement or amplicon thereof is released from the array feature by physical means.
• methods for inducing physical release include denaturing double stranded nucleic acid molecules.
• Another method for releasing the extended capture probes is to use a solution that interferes with the hydrogen bonds of the double stranded molecules.
• the extended capture probe is released by applying heated water such as water or buffer of at least 85° C., e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99° C.
• a formamide solution can be used to destabilize the interaction between nucleic acid molecules to release the extended capture probe from the array feature.
• an “analyte capture agent” refers to an agent that interacts with an analyte (e.g., an analyte in a sample) and with a capture probe (e.g., a capture probe attached to a substrate) to identify the analyte.
• the analyte capture agent includes an analyte binding moiety and a capture agent barcode domain.
• FIG. 9 is a schematic diagram of an exemplary analyte capture agent 902 comprised of an analyte binding moiety 904 and a capture agent barcode domain 908 .
• An analyte binding moiety 904 is a molecule capable of binding to an analyte 906 and interacting with a spatially-barcoded capture probe.
• the analyte binding moiety can bind to the analyte 906 with high affinity and/or with high specificity.
• the analyte capture agent can include a capture agent barcode domain 908 , a nucleotide sequence (e.g., an oligonucleotide), which can hybridize to at least a portion or an entirety of a capture domain of a capture probe.
• the analyte binding moiety 904 can include a polypeptide and/or an aptamer (e.g., an oligonucleotide or peptide molecule that binds to a specific target analyte).
• the analyte binding moiety 904 can include an antibody or antibody fragment (e.g., an antigen-binding fragment).
• analyte binding moiety refers to a molecule or moiety capable of binding to a macromolecular constituent (e.g., an analyte, e.g., a biological analyte).
• the analyte binding moiety of the analyte capture agent that binds to a biological analyte can include, but is not limited to, an antibody, or an epitope binding fragment thereof, a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi-specific antibody, a bi-specific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold, or any combination thereof.
• the analyte binding moiety can bind to the macromolecular constituent (e.g., analyte) with high affinity and/or with high specificity.
• the analyte binding moiety can include a nucleotide sequence (e.g., an oligonucleotide), which can correspond to at least a portion or an entirety of the analyte binding moiety.
• the analyte binding moiety can include a polypeptide and/or an aptamer (e.g., a polypeptide and/or an aptamer that binds to a specific target molecule, e.g., an analyte).
• the analyte binding moiety can include an antibody or antibody fragment (e.g., an antigen-binding fragment) that binds to a specific analyte (e.g., a polypeptide).
• analyte capture agents are capable of binding to analytes present inside a cell.
• analyte capture agents are capable of binding to cell surface analytes that can include, without limitation, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, an extracellular matrix protein, a posttranslational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation or lipidation) state of a cell surface protein, a gap junction, and an adherens junction.
• a posttranslational modification e.g
• the analyte capture agents are capable of binding to cell surface analytes that are post-translationally modified.
• analyte capture agents can be specific for cell surface analytes based on a given state of posttranslational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation or lipidation), such that a cell surface analyte profile can include posttranslational modification information of one or more analytes.
• the analyte capture agent includes a capture agent barcode domain that is conjugated or otherwise attached to the analyte binding moiety. In some embodiments, the capture agent barcode domain is covalently-linked to the analyte binding moiety. In some embodiments, a capture agent barcode domain is a nucleic acid sequence. In some embodiments, a capture agent barcode domain includes an analyte binding moiety barcode and an analyte capture sequence.
• analyte binding moiety barcode refers to a barcode that is associated with or otherwise identifies the analyte binding moiety. In some embodiments, by identifying an analyte binding moiety by identifying its associated analyte binding moiety barcode, the analyte to which the analyte binding moiety binds can also be identified.
• An analyte binding moiety barcode can be a nucleic acid sequence of a given length and/or sequence that is associated with the analyte binding moiety.
• An analyte binding moiety barcode can generally include any of the variety of aspects of barcodes described herein.
• an analyte capture agent that is specific to one type of analyte can have coupled thereto a first capture agent barcode domain (e.g., that includes a first analyte binding moiety barcode), while an analyte capture agent that is specific to a different analyte can have a different capture agent barcode domain (e.g., that includes a second barcode analyte binding moiety barcode) coupled thereto.
• a capture agent barcode domain can include an analyte binding moiety barcode that permits identification of the analyte binding moiety to which the capture agent barcode domain is coupled.
• the analyte capture agents can include analyte binding moieties with capture agent barcode domains attached to them.
• an analyte capture agent can include a first analyte binding moiety (e.g., an antibody that binds to an analyte, e.g., a first cell surface feature) having associated with it a capture agent barcode domain that includes a first analyte binding moiety barcode.
• the capture agent barcode domain of an analyte capture agent includes an analyte capture sequence.
• analyte capture sequence refers to region or moiety of configured to hybridize to, bind to, couple to, or otherwise interact with a capture domain of a capture probe.
• an analyte capture sequence includes a nucleic acid sequence that is complementary to or substantially complementary to the capture domain of a capture probe such that the analyte capture sequence hybridizes to the capture domain of the capture probe.
• an analyte capture sequence comprises a poly(A) nucleic acid sequence that hybridizes to a capture domain that comprises a poly(T) nucleic acid sequence.
• an analyte capture sequence comprises a poly(T) nucleic acid sequence that hybridizes to a capture domain that comprises a poly(A) nucleic acid sequence. In some embodiments, an analyte capture sequence comprises a non-homopolymeric nucleic acid sequence that hybridizes to a capture domain that comprises a non-homopolymeric nucleic acid sequence that is complementary (or substantially complementary) to the non-homopolymeric nucleic acid sequence of the analyte capture region.
• the capture agent barcode domain can be directly coupled to the analyte binding moiety, or they can be attached to a bead, molecular lattice, e.g., a linear, globular, cross-slinked, or other polymer, or other framework that is attached or otherwise associated with the analyte binding moiety, which allows attachment of multiple capture agent barcode domains to a single analyte binding moiety.
• Attachment (coupling) of the capture agent barcode domains to the analyte binding moieties can be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments.
• capture agent barcode domains can be covalently attached to a portion of the antibody or antigen-binding fragment using chemical conjugation techniques (e.g., Lightning-Link® antibody labelling kits available from Innova Biosciences).
• chemical conjugation techniques e.g., Lightning-Link® antibody labelling kits available from Innova Biosciences.
• a capture agent barcode domain can be coupled to an antibody or antigen-binding fragment using non-covalent attachment mechanisms (e.g., using biotinylated antibodies and oligonucleotides or beads that include one or more biotinylated linker, coupled to oligonucleotides with an avidin or streptavidin linker.)
• Antibody and oligonucleotide biotinylation techniques can be used, and are described for example in Fang et al., Nucleic Acids Res. (2003), 31(2): 708-715, the entire contents of which are incorporated by reference herein.
• protein and peptide biotinylation techniques have been developed and can be used, and are described for example in U.S.
• click reaction chemistry such as a methyltetrazine-PEG5-NHS ester reaction, a TCO-PEG4-NHS ester reaction, or the like, can be used to couple capture agent barcode domains to analyte binding moieties.
• the reactive moiety on the analyte binding moiety can also include amine for targeting aldehydes, amine for targeting maleimide (e.g., free thiols), azide for targeting click chemistry compounds (e.g., alkynes), biotin for targeting streptavidin, phosphates for targeting EDC, which in turn targets active ester (e.g., NH2).
• the reactive moiety on the analyte binding moiety can be a chemical compound or group that binds to the reactive moiety on the analyte binding moiety.
• exemplary strategies to conjugate the analyte binding moiety to the capture agent barcode domain include the use of commercial kits (e.g., Solulink, Thunder link), conjugation of mild reduction of hinge region and maleimide labelling, stain-promoted click chemistry reaction to labeled amides (e.g., copper-free), and conjugation of periodate oxidation of sugar chain and amine conjugation.
• the analyte binding moiety is an antibody
• the antibody can be modified prior to or contemporaneously with conjugation of the oligonucleotide.
• the antibody can be glycosylated with a substrate-permissive mutant of ⁇ -1,4-galactosyltransferase, GalT (Y289L) and azide-bearing uridine diphosphate-N-acetylgalactosamine analog uridine diphosphate-GalNAz.
• the modified antibody can be conjugated to an oligonucleotide with a dibenzocyclooctyne-PEG4-NHS group.
• certain steps e.g., COOH activation (e.g., EDC) and homobifunctional cross linkers
• COOH activation e.g., EDC
• homobifunctional cross linkers can be avoided to prevent the analyte binding moieties from conjugating to themselves.
• the analyte capture agent e.g., analyte binding moiety coupled to an oligonucleotide
• the analyte capture agent can be delivered into the cell, e.g., by transfection (e.g., using transfectamine, cationic polymers, calcium phosphate or electroporation), by transduction (e.g., using a bacteriophage or recombinant viral vector), by mechanical delivery (e.g., magnetic beads), by lipid (e.g., 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)), or by transporter proteins.
• transfection e.g., using transfectamine, cationic polymers, calcium phosphate or electroporation
• transduction e.g., using a bacteriophage or recombinant viral vector
• mechanical delivery e.g., magnetic beads
• lipid e.g., 1,2-Dioleoyl-
• An analyte capture agent can be delivered into a cell using exosomes.
• a first cell can be generated that releases exosomes comprising an analyte capture agent.
• An analyte capture agent can be attached to an exosome membrane.
• An analyte capture agent can be contained within the cytosol of an exosome. Released exosomes can be harvested and provided to a second cell, thereby delivering the analyte capture agent into the second cell.
• An analyte capture agent can be releasable from an exosome membrane before, during, or after delivery into a cell.
• the cell is permeabilized to allow the analyte capture agent to couple with intracellular cellular constituents (such as, without limitation, intracellular proteins, metabolites and nuclear membrane proteins).
• intracellular cellular constituents such as, without limitation, intracellular proteins, metabolites and nuclear membrane proteins.
• analyte capture agents can be used to analyze intracellular constituents as described herein.
• the capture agent barcode domain coupled to an analyte capture agent can include modifications that render it non-extendable by a polymerase.
• the capture agent barcode domain when binding to a capture domain of a capture probe or nucleic acid in a sample for a primer extension reaction, the capture agent barcode domain can serve as a template, not a primer.
• the capture agent barcode domain also includes a barcode (e.g., an analyte binding moiety barcode)
• such a design can increase the efficiency of molecular barcoding by increasing the affinity between the capture agent barcode domain and unbarcoded sample nucleic acids, and eliminate the potential formation of adaptor artifacts.
• the capture agent barcode domain can include a random N-mer sequence that is capped with modifications that render it non-extendable by a polymerase.
• the composition of the random N-mer sequence can be designed to maximize the binding efficiency to free, unbarcoded ssDNA molecules.
• the design can include a random sequence composition with a higher GC content, a partial random sequence with fixed G or C at specific positions, the use of guanosines, the use of locked nucleic acids, or any combination thereof.
• a modification for blocking primer extension by a polymerase can be a carbon spacer group of different lengths or a dideoxynucleotide.
• the modification can be an abasic site that has an apurine or apyrimidine structure, a base analog, or an analogue of a phosphate backbone, such as a backbone of N-(2-aminoethyl)-glycine linked by amide bonds, tetrahydrofuran, or 1′, 2′-Dideoxyribose.
• the modification can also be a uracil base, 2′OMe modified RNA, C3-18 spacers (e.g., structures with 3-18 consecutive carbon atoms, such as C3 spacer), ethylene glycol multimer spacers (e.g., spacer 18 (hexa-ethyleneglycol spacer), biotin, di-deoxynucleotide triphosphate, ethylene glycol, amine, or phosphate.
• C3-18 spacers e.g., structures with 3-18 consecutive carbon atoms, such as C3 spacer
• ethylene glycol multimer spacers e.g., spacer 18 (hexa-ethyleneglycol spacer)
• biotin di-deoxynucleotide triphosphate
• ethylene glycol, amine, or phosphate e.g., hexa-ethyleneglycol spacer
• the capture agent barcode domain coupled to the analyte binding moiety includes a cleavable domain.
• the capture agent barcode domain can be cleaved and collected for downstream analysis according to the methods as described herein.
• the cleavable domain of the capture agent barcode domain includes a U-excising element that allows the species to release from the bead.
• the U-excising element can include a single-stranded DNA (ssDNA) sequence that contains at least one uracil.
• the species can be attached to a bead via the ssDNA sequence.
• the species can be released by a combination of uracil-DNA glycosylase (e.g., to remove the uracil) and an endonuclease (e.g., to induce an ssDNA break). If the endonuclease generates a 5′ phosphate group from the cleavage, then additional enzyme treatment can be included in downstream processing to eliminate the phosphate group, e.g., prior to ligation of additional sequencing handle elements, e.g., Illumina full P5 sequence, partial P5 sequence, full R1 sequence, and/or partial R1 sequence.
• an analyte binding moiety of an analyte capture agent includes one or more antibodies or antigen binding fragments thereof.
• the antibodies or antigen binding fragments including the analyte binding moiety can specifically bind to a target analyte.
• the analyte is a protein (e.g., a protein on a surface of the biological sample (e.g., a cell) or an intracellular protein).
• a plurality of analyte capture agents comprising a plurality of analyte binding moieties bind a plurality of analytes present in a biological sample.
• the plurality of analytes includes a single species of analyte (e.g., a single species of polypeptide). In some embodiments in which the plurality of analytes includes a single species of analyte, the analyte binding moieties of the plurality of analyte capture agents are the same.
• the analyte binding moieties of the plurality of analyte capture agents are the different (e.g., members of the plurality of analyte capture agents can have two or more species of analyte binding moieties, wherein each of the two or more species of analyte binding moieties binds a single species of analyte, e.g., at different binding sites).
• the plurality of analytes includes multiple different species of analyte (e.g., multiple different species of polypeptides).
• multiple different species of analytes from the biological sample can be subsequently associated with the one or more physical properties of the biological sample.
• the multiple different species of analytes can be associated with locations of the analytes in the biological sample.
• Such information e.g., proteomic information when the analyte binding moiety(ies) recognizes a polypeptide(s)
• can be used in association with other spatial information e.g., genetic information from the biological sample, such as DNA sequence information, transcriptome information (i.e., sequences of transcripts), or both).
• a cell surface protein of a cell can be associated with one or more physical properties of the cell (e.g., a shape, size, activity, or a type of the cell).
• the one or more physical properties can be characterized by imaging the cell.
• the cell can be bound by an analyte capture agent comprising an analyte binding moiety that binds to the cell surface protein and an analyte binding moiety barcode that identifies that analyte binding moiety, and the cell can be subjected to spatial analysis (e.g., any of the variety of spatial analysis methods described herein).
• the analyte capture agent bound to the cell surface protein can be bound to a capture probe (e.g., a capture probe on an array), which capture probe includes a capture domain that interacts with an analyte capture sequence present on the capture agent barcode domain of the analyte capture agent.
• All or part of the capture agent barcode domain can be copied with a polymerase using a 3′ end of the capture domain as a priming site, generating an extended capture probe that includes the all or part of the capture probe (including a spatial barcode present on the capture probe) and a copy of the analyte binding moiety barcode.
• the spatial array with the extended capture probe(s) can be contacted with a sample, where the analyte capture agent(s) associated with the spatial array capture the target analyte(s).
• the analyte capture agent(s) containing the extended capture probe(s), which includes the spatial barcode(s) of the capture probe(s) and the analyte binding moiety barcode(s) can then be denatured from the capture probe(s) of the spatial array. This allows the spatial array to be reused.
• the sample can be dissociated into non-aggregated cells (e.g. single cells) and analyzed by the single cell/droplet methods described herein.
• the extended capture probe can be sequenced to obtain a nucleic acid sequence, in which the spatial barcode of the capture probe is associated with the analyte binding moiety barcode of the analyte capture agent.
• the nucleic acid sequence of the extended capture probe can thus be associated with the analyte (e.g., cell surface protein), and in turn, with the one or more physical properties of the cell (e.g., a shape or cell type).
• the nucleic acid sequence of the extended capture probe can be associated with an intracellular analyte of a nearby cell, where the intracellular analyte was released using any of the cell permeabilization or analyte migration techniques described herein.
• the capture agent barcode domains released from the analyte capture agents can then be subjected to sequence analysis to identify which analyte capture agents were bound to analytes.
• sequence analysis to identify which analyte capture agents were bound to analytes.
• an analyte profile can be created for a biological sample.
• Profiles of individual cells or populations of cells can be compared to profiles from other cells, e.g., ‘normal’ cells, to identify variations in analytes, which can provide diagnostically relevant information.
• these profiles can be useful in the diagnosis of a variety of disorders that are characterized by variations in cell surface receptors, such as cancer and other disorders.
• FIG. 10 is a schematic diagram depicting an exemplary interaction between a feature-immobilized capture probe 1024 and an analyte capture agent 1026 .
• the feature-immobilized capture probe 1024 can include a spatial barcode 1008 as well as one or more functional sequences 1006 and 1010 , as described elsewhere herein.
• the capture probe can also include a capture domain 1012 that is capable of binding to an analyte capture agent 1026 .
• the analyte capture agent 1026 can include a functional sequence 1018 , capture agent barcode domain 1016 , and an analyte capture sequence 1014 that is capable of binding to the capture domain 1012 of the capture probe 1024 .
• the analyte capture agent can also include a linker 1020 that allows the capture agent barcode domain 1016 to couple to the analyte binding moiety 1022 .
• the methods are used to identify immune cell profiles.
• Immune cells express various adaptive immunological receptors relating to immune function, such as T cell receptors (TCRs) and B cell receptors (BCRs).
• TCRs T cell receptors
• BCRs B cell receptors
• T cell receptors and B cell receptors play a part in the immune response by specifically recognizing and binding to antigens and aiding in their destruction.
• the T cell receptor is a molecule found on the surface of T cells that is generally responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules.
• MHC major histocompatibility complex
• the TCR is generally a heterodimer of two chains, each of which is a member of the immunoglobulin superfamily, possessing an N-terminal variable (V) domain, and a C terminal constant domain.
• V N-terminal variable
• V N-terminal variable
• C terminal constant domain In humans, in 95% of T cells, the TCR consists of an alpha ( ⁇ ) and beta ( ⁇ ) chain, whereas in 5% of T cells, the TCR consists of gamma and delta ( ⁇ / ⁇ ) chains. This ratio can change during ontogeny and in diseased states as well as in different species.
• Each of the two chains of a TCR contains multiple copies of gene segments—a variable ‘V’ gene segment, a diversity ‘D’ gene segment, and a joining ‘J’ gene segment.
• the TCR alpha chain (TCRa) is generated by recombination of V and J segments, while the beta chain (TCRb) is generated by recombination of V, D, and J segments.
• generation of the TCR gamma chain involves recombination of V and J gene segments, while generation of the TCR delta chain occurs by recombination of V, D, and J gene segments.
• Complementarity determining regions e.g., CDR1, CDR2, and CDR3, or hypervariable regions, are sequences in the variable domains of antigen receptors (e.g., T cell receptor and immunoglobulin) that can complement an antigen.
• antigen receptors e.g., T cell receptor and immunoglobulin
• Most of the diversity of CDRs is found in CDR3, with the diversity being generated by somatic recombination events during the development of T lymphocytes.
• a unique nucleotide sequence that arises during the gene arrangement process can be referred to as a clonotype.
• the B cell receptor is a molecule found on the surface of B cells.
• the antigen binding portion of a BCR is composed of a membrane-bound antibody that, like most antibodies (e.g., immunoglobulins), has a unique and randomly determined antigen-binding site.
• the antigen binding portion of a BCR includes membrane-bound immunoglobulin molecule of one isotype (e.g., IgD, IgM, IgA, IgG, or IgE).
• the BCR is composed of two genes IgH and IgK (or IgL) coding for antibody heavy and light chains. Immunoglobulins are formed by recombination among gene segments, sequence diversification at the junctions of these segments, and point mutations throughout the gene. Each heavy chain gene contains multiple copies of three different gene segments—a variable ‘V’ gene segment, a diversity ‘D’ gene segment, and a joining ‘J’ gene segment. Each light chain gene contains multiple copies of two different gene segments for the variable region of the protein—a variable ‘V’ gene segment and a joining ‘J’ gene segment.
• the recombination can generate a molecule with one of each of the V, D, and J segments. Furthermore, several bases can be deleted and others added (called N and P nucleotides) at each of the two junctions, thereby generating further diversity.
• N and P nucleotides are added at each of the two junctions, thereby generating further diversity.
• activated B cells undergo the process of isotype switching.
• Antibodies with the same variable segments can have different forms (isotypes) depending on the constant segment. Whereas all na ⁇ ve B cells express IgM (or IgD), activated B cells mostly express IgG but also IgM, IgA and IgE. This expression switching from IgM (and/or IgD) to IgG, IgA, or IgE occurs through a recombination event causing one cell to specialize in producing a specific isotype.
• a unique nucleotide sequence that arises during the gene arrangement process can similarly be referred to as a clonotype.
• Certain methods described herein are utilized to analyze the various sequences of TCRs and BCRs from immune cells, for example, various clonotypes.
• the methods are used to analyze the sequence of a TCR alpha chain, a TCR beta chain, a TCR delta chain, a TCR gamma chain, or any fragment thereof (e.g., variable regions including V(D)J or VJ regions, constant regions, transmembrane regions, fragments thereof, combinations thereof, and combinations of fragments thereof).
• the methods described herein can be used to analyze the sequence of a B cell receptor heavy chain, B cell receptor light chain, or any fragment thereof (e.g., variable regions including V(D)J or VJ regions, constant regions, transmembrane regions, fragments thereof, combinations thereof, and combinations of fragments thereof).
• primer sequences useful in any of the various operations for attaching barcode sequences and/or amplification reactions can include gene specific sequences which target genes or regions of genes of immune cell proteins, for example immune receptors.
• gene sequences include, but are not limited to, sequences of various T cell receptor alpha variable genes (TRAV genes), T cell receptor alpha joining genes (TRAJ genes), T cell receptor alpha constant genes (TRAC genes), T cell receptor beta variable genes (TRBV genes), T cell receptor beta diversity genes (TRBD genes), T cell receptor beta joining genes (TRBJ genes), T cell receptor beta constant genes (TRBC genes), T cell receptor gamma variable genes (TRGV genes), T cell receptor gamma joining genes (TRGJ genes), T cell receptor gamma constant genes (TRGC genes), T cell receptor delta variable genes (TRDV genes), T cell receptor delta diversity genes (TRDD genes), T cell receptor delta joining genes (TRDJ genes), and T cell receptor delta constant genes (TRDC genes).
• TRAV genes T cell receptor alpha variable genes
• TRAJ genes
• the analyte binding moiety is based on the Major Histocompatibility Complex (MHC) class I or class II.
• MHC Major Histocompatibility Complex
• the analyte binding moiety is an MHC multimer including, without limitation, MHC dextramers, MHC tetramers, and MHC pentamers (see, for example, U.S. Patent Application Publication Nos. US 2018/0180601 and US 2017/0343545, the entire contents of each of which are incorporated herein by reference.
• MHCs e.g., a soluble MHC monomer molecule
• MHCs can be used as analyte binding moieties of analyte capture agents that are coupled to capture agent barcode domains that include an analyte binding moiety barcode that identifies its associated MHC (and, thus, for example, the MHC's TCR binding partner).
• MHCs are used to analyze one or more cell-surface features of a T-cell, such as a TCR.
• multiple MHCs are associated together in a larger complex (MHC multimer) to improve binding affinity of MHCs to TCRs via multiple ligand binding synergies.
• FIGS. 11 A, 11 B, and 11 C are schematics illustrating how streptavidin cell tags can be utilized in an array-based system to produce a spatially-barcoded cell or cellular contents.
• peptide-bound major histocompatibility complex pMHCs
• pMHCs can be individually associated with biotin and bound to a streptavidin moiety such that the streptavidin moiety comprises multiple pMHC moieties.
• Each of these moieties can bind to a TCR such that the streptavidin binds to a target T-cell via multiple MCH/TCR binding interactions. Multiple interactions synergize and can substantially improve binding affinity.
• a capture agent barcode domain 1101 can be modified with streptavidin 1102 and contacted with multiple molecules of biotinylated MHC 1103 (such as a pMHC) such that the biotinylated MHC 1103 molecules are coupled with the streptavidin conjugated capture agent barcode domain 1101 .
• biotinylated MHC 1103 such as a pMHC
• the result is a barcoded MHC multimer complex 1105 .
• the capture agent barcode domain sequence 1101 can identify the MHC as its associated label and also includes optional functional sequences such as sequences for hybridization with other oligonucleotides. As shown in FIG.
• one example oligonucleotide is capture probe 1106 that comprises a complementary sequence (e.g., rGrGrG corresponding to C C C), a barcode sequence and other functional sequences, such as, for example, a UMI, an adapter sequence (e.g., comprising a sequencing primer sequence (e.g., R1 or a partial R1 (“pR1”)), a flow cell attachment sequence (e.g., P5 or P7 or partial sequences thereof)), etc.
• capture probe 1106 may at first be associated with a feature (e.g., a gel bead) and released from the feature.
• capture probe 1106 can hybridize with a capture agent barcode domain 1101 of the MHC-oligonucleotide complex 1105 .
• the hybridized oligonucleotides (Spacer C C C and Spacer rGrGrG) can then be extended in primer extension reactions such that constructs comprising sequences that correspond to each of the two spatial barcode sequences (the spatial barcode associated with the capture probe, and the barcode associated with the MHC-oligonucleotide complex) are generated.
• one or both of these corresponding sequences may be a complement of the original sequence in capture probe 1106 or capture agent barcode domain 1101 .
• the capture probe and the capture agent barcode domain are ligated together.
• the resulting constructs can be optionally further processed (e.g., to add any additional sequences and/or for clean-up) and subjected to sequencing.
• a sequence derived from the capture probe 1106 spatial barcode sequence may be used to identify a feature and the sequence derived from spatial barcode sequence on the capture agent barcode domain 1101 may be used to identify the particular peptide MHC complex 1104 bound on the surface of the cell (e.g., when using MHC-peptide libraries for screening immune cells or immune cell populations).
• the substrate functions as a support for direct or indirect attachment of capture probes to features of the array.
• a substrate e.g., the same substrate or a different substrate
• a substrate can be used to provide support to a biological sample, particularly, for example, a thin tissue section.
• a “substrate” is a support that is insoluble in aqueous liquid and which allows for positioning of biological samples, analytes, features, and/or capture probes on the substrate.
• a substrate can be any suitable support material.
• Exemplary substrates include, but are not limited to, glass, modified and/or functionalized glass, hydrogels, films, membranes, plastics (including e.g., acrylics, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonTM, cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers, such as polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polypropylene, polyethylene and polycarbonate.
• plastics including e.g., acrylics, polystyrene, copolymers of styrene and other materials,
• the substrate can also correspond to a flow cell.
• Flow cells can be formed of any of the foregoing materials, and can include channels that permit reagents, solvents, features, and molecules to pass through the cell.
• polystyrene is a hydrophobic material suitable for binding negatively charged macromolecules because it normally contains few hydrophilic groups.
• nucleic acids immobilized on glass slides by increasing the hydrophobicity of the glass surface the nucleic acid immobilization can be increased.
• Such an enhancement can permit a relatively more densely packed formation (e.g., provide improved specificity and resolution).
• a substrate is coated with a surface treatment such as poly(L)-lysine. Additionally or alternatively, the substrate can be treated by silanation, e.g. with epoxy-silane, amino-silane, and/or by a treatment with polyacrylamide.
• the substrate can generally have any suitable form or format.
• the substrate can be flat, curved, e.g. convexly or concavely curved towards the area where the interaction between a biological sample, e.g. tissue sample, and the substrate takes place.
• the substrate is a flat, e.g., planar, chip or slide.
• the substrate can contain one or more patterned surfaces within the substrate (e.g., channels, wells, projections, ridges, divots, etc.).
• a substrate can be of any desired shape.
• a substrate can be typically a thin, flat shape (e.g., a square or a rectangle).
• a substrate structure has rounded corners (e.g., for increased safety or robustness).
• a substrate structure has one or more cut-off corners (e.g., for use with a slide clamp or cross-table).
• the substrate structure can be any appropriate type of support having a flat surface (e.g., a chip or a slide such as a microscope slide).
• Substrates can optionally include various structures such as, but not limited to, projections, ridges, and channels.
• a substrate can be micropatterned to limit lateral diffusion (e.g., to prevent overlap of spatial barcodes).
• a substrate modified with such structures can be modified to allow association of analytes, features (e.g., beads), or probes at individual sites.
• the sites where a substrate is modified with various structures can be contiguous or non-contiguous with other sites.
• the surface of a substrate can be modified so that discrete sites are formed that can only have or accommodate a single feature. In some embodiments, the surface of a substrate can be modified so that features adhere to random sites.
• the surface of a substrate is modified to contain one or more wells, using techniques such as (but not limited to) stamping techniques, microetching techniques, and molding techniques.
• the substrate in which a substrate includes one or more wells, can be a concavity slide or cavity slide.
• wells can be formed by one or more shallow depressions on the surface of the substrate.
• the wells can be formed by attaching a cassette (e.g., a cassette containing one or more chambers) to a surface of the substrate structure.
• the structures of a substrate can each bear a different capture probe.
• Different capture probes attached to each structure can be identified according to the locations of the structures in or on the surface of the substrate.
• Exemplary substrates include arrays in which separate structures are located on the substrate including, for example, those having wells that accommodate features.
• a substrate includes one or more markings on a surface of the substrate, e.g., to provide guidance for correlating spatial information with the characterization of the analyte of interest.
• a substrate can be marked with a grid of lines (e.g., to allow the size of objects seen under magnification to be easily estimated and/or to provide reference areas for counting objects).
• fiducial markers can be included on the substrate. Such markings can be made using techniques including, but not limited to, printing, sand-blasting, and depositing on the surface.
• the structures can include physically altered sites.
• a substrate modified with various structures can include physical properties, including, but not limited to, physical configurations, magnetic or compressive forces, chemically functionalized sites, chemically altered sites, and/or electrostatically altered sites.
• the structures are applied in a pattern.
• the structures can be randomly distributed.
• a substrate is treated in order to minimize or reduce non-specific analyte hybridization within or between features.
• treatment can include coating the substrate with a hydrogel, film, and/or membrane that creates a physical barrier to non-specific hybridization.
• Any suitable hydrogel can be used.
• hydrogel matrices prepared according to the methods set forth in U.S. Pat. Nos. 6,391,937, 9,512,422, and 9,889,422, and U.S. Patent Application Publication Nos. U.S. 2017/0253918 and U.S. 2018/0052081 can be used. The entire contents of each of the foregoing documents are incorporated herein by reference.
• Treatment can include adding a functional group that is reactive or capable of being activated such that it becomes reactive after receiving a stimulus (e.g., photoreactive).
• Treatment can include treating with polymers having one or more physical properties (e.g., mechanical, electrical, magnetic, and/or thermal) that minimize non-specific binding (e.g., that activate a substrate at certain locations to allow analyte hybridization at those locations).
• the substrate e.g., a bead or a feature on an array
• the substrate can include tens to hundreds of thousands or millions of individual oligonucleotide molecules (e.g., at least about 10,000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000, or 10,000,000,000 oligonucleotide molecules).
• the surface of the substrate is coated with a cell-permissive coating to allow adherence of live cells.
• a “cell-permissive coating” is a coating that allows or helps cells to maintain cell viability (e.g., remain viable) on the substrate.
• a cell-permissive coating can enhance cell attachment, cell growth, and/or cell differentiation, e.g., a cell-permissive coating can provide nutrients to the live cells.
• a cell-permissive coating can include a biological material and/or a synthetic material.
• Non-limiting examples of a cell-permissive coating include coatings that feature one or more extracellular matrix (ECM) components (e.g., proteoglycans and fibrous proteins such as collagen, elastin, fibronectin and laminin), poly-lysine, poly(L)-ornithine, and/or a biocompatible silicone (e.g., CYTOSOFT®).
• ECM extracellular matrix
• a cell-permissive coating that includes one or more extracellular matrix components can include collagen Type I, collagen Type II, collagen Type IV, elastin, fibronectin, laminin, and/or vitronectin.
• the cell-permissive coating includes a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g., MATRIGEL® (matrix derived from mouse tumor rich in laminin, collagen, and other ECM proteins)).
• EHS Engelbreth-Holm-Swarm
• the cell-permissive coating includes collagen.
• a cell-permissive coating can be used to culture adherent cells on a spatially-barcoded array, or to maintain cell viability of a tissue sample or section while in contact with a spatially-barcoded array.
• oligonucleotides within the gel can attach to the substrate.
• a gel e.g., a hydrogel or gel matrix
• oligonucleotides within the gel can attach to the substrate.
• the terms “hydrogel” and “hydrogel matrix” are used interchangeably herein to refer to a macromolecular polymer gel including a network. Within the network, some polymer chains can optionally be cross-linked, although cross-linking does not always occur.
• a hydrogel can include hydrogel subunits.
• a “hydrogel subunit” is a hydrophilic monomer, a molecular precursor, or a polymer that can be polymerized (e.g., cross-linked) to form a three-dimensional (3D) hydrogel network.
• the hydrogel subunits can include any convenient hydrogel subunits, such as, but not limited to, acrylamide, bis-acrylamide, polyacrylamide and derivatives thereof, poly(ethylene glycol) and derivatives thereof (e.g.
• PEG-acrylate PEG-DA
• PEG-RGD gelatin-methacryloyl
• GelMA gelatin-methacryloyl
• Methacrylated hyaluronic acid MeHA
• polyaliphatic polyurethanes polyether polyurethanes
• polyester polyurethanes polyethylene copolymers
• polyamides polyvinyl alcohols, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone
• polyacrylamide poly(hydroxyethyl acrylate)
• collagen hyaluronic acid
• chitosan dextran
• agarose gelatin
• gelatin alginate
• protein polymers methylcellulose, and the like, and combinations thereof.
• a hydrogel includes a hybrid material, e.g., the hydrogel material includes elements of both synthetic and natural polymers.
• the hydrogel material includes elements of both synthetic and natural polymers. Examples of suitable hydrogels are described, for example, in U.S. Pat. Nos. 6,391,937, 9,512,422, and 9,889,422, and in U.S. Patent Application Publication Nos. 2017/0253918, 2018/0052081 and 2010/0055733, the entire contents of each of which are incorporated herein by reference.
• cross-linkers and/or initiators are added to hydrogel subunits.
• cross-linkers include, without limitation, bis-acrylamide and diazirine.
• initiators include, without limitation, azobisisobutyronitrile (AIBN), riboflavin, and L-arginine. Inclusion of cross-linkers and/or initiators can lead to increased covalent bonding between interacting biological macromolecules in later polymerization steps.
• hydrogels can have a colloidal structure, such as agarose, or a polymer mesh structure, such as gelatin.
• hydrogel subunits are polymerized (e.g., undergo “formation”) covalently or physically cross-linked, to form a hydrogel network.
• hydrogel subunits can be polymerized by any method including, but not limited to, thermal crosslinking, chemical crosslinking, physical crosslinking, ionic crosslinking, photo-crosslinking, irradiative crosslinking (e.g., x-ray, electron beam), and combinations thereof. Techniques such as lithographic photopolymerization can also be used to form hydrogels.
• Polymerization methods for hydrogel subunits can be selected to form hydrogels with different properties (e.g., pore size, swelling properties, biodegradability, conduction, transparency, and/or permeability of the hydrogel).
• a hydrogel can include pores of sufficient size to allow the passage of macromolecules, (e.g., nucleic acids, proteins, chromatin, metabolites, gRNA, antibodies, carbohydrates, peptides, metabolites, and/or small molecules) into the sample (e.g., tissue section).
• macromolecules e.g., nucleic acids, proteins, chromatin, metabolites, gRNA, antibodies, carbohydrates, peptides, metabolites, and/or small molecules
• a fixative/hydrogel composition can be prepared that includes a concentration of hydrogel subunits that allows the passage of such biological macromolecules.
• the hydrogel can form the substrate.
• the substrate includes a hydrogel and one or more second materials.
• the hydrogel is placed on top of one or more second materials.
• the hydrogel can be pre-formed and then placed on top of, underneath, or in any other configuration with one or more second materials.
• hydrogel formation occurs after contacting one or more second materials during formation of the substrate. Hydrogel formation can also occur within a structure (e.g., wells, ridges, projections, and/or markings) located on a substrate.
• hydrogel formation on a substrate occurs before, contemporaneously with, or after features (e.g., beads) are attached to the substrate.
• features e.g., beads
• hydrogel formation can be performed on the substrate already containing the capture probes.
• hydrogel formation occurs within a biological sample.
• a biological sample e.g., tissue section
• hydrogel subunits are infused into the biological sample, and polymerization of the hydrogel is initiated by an external or internal stimulus.
• functionalization chemistry in embodiments in which a hydrogel is formed within a biological sample, functionalization chemistry can be used.
• functionalization chemistry includes hydrogel-tissue chemistry (HTC).
• HTC hydrogel-tissue chemistry
• Any hydrogel-tissue backbone (e.g., synthetic or native) suitable for HTC can be used for anchoring biological marcomolecules and modulating functionalization.
• Non-limiting examples of methods using HTC backbone variants include CLARITY, PACT, ExM, SWITCH and ePACT.
• hydrogel formation within a biological sample is permanent.
• biological macromolecules can permanently adhere to the hydrogel allowing multiple rounds of interrogation.
• hydrogel formation within a biological sample is reversible.
• additional reagents are added to the hydrogel subunits before, contemporaneously with, and/or after polymerization.
• additional reagents can include but are not limited to oligonucleotides (e.g., capture probes), endonucleases to fragment DNA, fragmentation buffer for DNA, DNA polymerase enzymes, dNTPs used to amplify the nucleic acid and to attach the barcode to the amplified fragments.
• Other enzymes can be used, including without limitation, RNA polymerase, transposase, ligase, proteinase K, and DNAse.
• Additional reagents can also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers, and switch oligonucleotides.
• optical labels are added to the hydrogel subunits before, contemporaneously with, and/or after polymerization.
• HTC reagents are added to the hydrogel before, contemporaneously with, and/or after polymerization.
• a cell labelling agent is added to the hydrogel before, contemporaneously with, and/or after polymerization.
• a cell-penetrating agent is added to the hydrogel before, contemporaneously with, and/or after polymerization.
• Hydrogels embedded within biological samples can be cleared using any suitable method.
• electrophoretic tissue clearing methods can be used to remove biological macromolecules from the hydrogel-embedded sample.
• a hydrogel-embedded sample is stored before or after clearing of hydrogel, in a medium (e.g., a mounting medium, methylcellulose, or other semi-solid mediums).
• a “conditionally removable coating” is a coating that can be removed from the surface of a substrate upon application of a releasing agent.
• a conditionally removable coating includes a hydrogel as described herein, e.g., a hydrogel including a polypeptide-based material.
• Non-limiting examples of a hydrogel featuring a polypeptide-based material include a synthetic peptide-based material featuring a combination of spider silk and a trans-membrane segment of human muscle L-type calcium channel (e.g., PEPGEL® (cell growth media)), an amphiphilic 16 residue peptide containing a repeating arginine-alanine-aspartate-alanine sequence (RADARADARADARADA) (SEQ ID NO: 22) (e.g., PURAMATRIX® (synthetic peptide hydrogels), EAK16 (AEAEAKAKAEAEAKAK) (SEQ ID NO: 23), KLD12 (KLDLKLDLKLDL) (SEQ ID NO: 24), and PGMATRIXTM (cell growth media).
• PEPGEL® cell growth media
• PURAMATRIX® synthetic peptide hydrogels
• EAK16 AEAEAKAKAEAEAKAK
• KLD12 KLDLKLDLKLDL
• SEQ ID NO: 24
• the hydrogel in the conditionally removable coating is a stimulus-responsive hydrogel.
• a stimulus-responsive hydrogel can undergo a gel-to-solution and/or gel-to-solid transition upon application of one or more external triggers (e.g., a releasing agent). See, e.g., Willner, Acc. Chem. Res. 50:657-658, 2017, which is incorporated herein by reference in its entirety.
• Non-limiting examples of a stimulus-responsive hydrogel include a thermoresponsive hydrogel, a pH-responsive hydrogel, a light-responsive hydrogel, a redox-responsive hydrogel, an analyte-responsive hydrogel, or a combination thereof.
• a stimulus-responsive hydrogel can be a multi-stimuli-responsive hydrogel.
• a “releasing agent” or “external trigger” is an agent that allows for the removal of a conditionally removable coating from a substrate when the releasing agent is applied to the conditionally removable coating.
• An external trigger or releasing agent can include physical triggers such as thermal, magnetic, ultrasonic, electrochemical, and/or light stimuli as well as chemical triggers such as pH, redox reactions, supramolecular complexes, and/or biocatalytically driven reactions. See e.g., Echeverria, et al., Gels (2016), 4, 54; doi:10.3390/gels4020054, which is incorporated herein by reference in its entirety.
• the type of “releasing agent” or “external trigger” can depend on the type of conditionally removable coating.
• a conditionally removable coating featuring a redox-responsive hydrogel can be removed upon application of a releasing agent that includes a reducing agent such as dithiothreitol (DTT).
• a pH-responsive hydrogel can be removed upon the application of a releasing agent that changes the pH.
• features are collectively positioned on a substrate.
• An “array” is a specific arrangement of a plurality of features that is either irregular or forms a regular pattern. Individual features in the array differ from one another based on their relative spatial locations. In general, at least two of the plurality of features in the array include a distinct capture probe (e.g., any of the examples of capture probes described herein).
• Arrays can be used to measure large numbers of analytes simultaneously.
• oligonucleotides are used, at least in part, to create an array.
• one or more copies of a single species of oligonucleotide e.g., capture probe
• a given feature in the array includes two or more species of oligonucleotides (e.g., capture probes).
• the two or more species of oligonucleotides (e.g., capture probes) attached directly or indirectly to a given feature on the array include a common (e.g., identical) spatial barcode.
• a “feature” is an entity that acts as a support or repository for various molecular entities used in sample analysis.
• features include, but are not limited to, a bead, a spot of any two- or three-dimensional geometry (e.g., an ink jet spot, a masked spot, a square on a grid), a well, and a hydrogel pad.
• features are directly or indirectly attached or fixed to a substrate.
• the features are not directly or indirectly attached or fixed to a substrate, but instead, for example, are disposed within an enclosed or partially enclosed three dimensional space (e.g., wells or divots).
• features that are formed from polymers and/or biopolymers that are jet printed, screen printed, or electrostatically deposited on a substrate can be used to form arrays.
• Jet printing of biopolymers is described, for example, in PCT Patent Application Publication No. WO 2014/085725. Jet printing of polymers is described, for example, in de Gans et al., Adv Mater. 16(3): 203-213 (2004). Methods for electrostatic deposition of polymers and biopolymers are described, for example, in Hoyer et al., Anal. Chem. 68(21): 3840-3844 (1996). The entire contents of each of the foregoing references are incorporated herein by reference.
• features are formed by metallic micro- or nanoparticles. Suitable methods for depositing such particles to form arrays are described, for example, in Lee et al., Beilstein J. Nanotechnol. 8: 1049-1055 (2017), the entire contents of which are incorporated herein by reference.
• features are formed by magnetic particles that are assembled on a substrate. Examples of such particles and methods for assembling arrays are described in Ye et al., Scientific Reports 6: 23145 (2016), the entire contents of which are incorporated herein by reference.
• features correspond to regions of a substrate in which one or more optical labels have been incorporated, and/or which have been altered by a process such as permanent photobleaching.
• Suitable substrates to implement features in this manner include a wide variety of polymers, for example. Methods for forming such features are described, for example, in Moshrefzadeh et al., Appl. Phys. Lett. 62: 16 (1993), the entire contents of which are incorporated herein by reference.
• features can correspond to colloidal particles assembled (e.g., via self-assembly) to form an array.
• colloidal particles are described for example in Sharma, Resonance 23(3): 263-275 (2016), the entire contents of which are incorporated herein by reference.
• features can be formed via spot-array photopolymerization of a monomer solution on a substrate.
• two-photon and three-photon polymerization can be used to fabricate features of relatively small (e.g., sub-micron) dimensions. Suitable methods for preparing features on a substrate in this manner are described for example in Nguyen et al., Materials Today 20(6): 314-322 (2017), the entire contents of which are incorporated herein by reference.
• features are directly or indirectly attached or fixed to a substrate that is liquid permeable. In some embodiments, features are directly or indirectly attached or fixed to a substrate that is biocompatible. In some embodiments, features are directly or indirectly attached or fixed to a substrate that is a hydrogel.
• FIG. 12 depicts an exemplary arrangement of barcoded features within an array. From left to right, FIG. 12 shows (L) a slide including six spatially-barcoded arrays, (C) an enlarged schematic of one of the six spatially-barcoded arrays, showing a grid of barcoded features in relation to a biological sample, and (R) an enlarged schematic of one section of an array, showing the specific identification of multiple features within the array (labelled as ID578, ID579, ID560, etc.).
• the term “bead array” refers to an array that includes a plurality of beads as the features in the array.
• the beads are attached to a substrate.
• the beads can optionally attach to a substrate such as a microscope slide and in proximity to a biological sample (e.g., a tissue section that includes cells).
• the beads can also be suspended in a solution and deposited on a surface (e.g., a membrane, a tissue section, or a substrate (e.g., a microscope slide)).
• arrays of beads on or within a substrate include beads located in wells such as the BeadChip array (available from Illumina Inc., San Diego, Calif.), arrays used in sequencing platforms from 454 LifeSciences (a subsidiary of Roche, Basel, Switzerland), and array used in sequencing platforms from Ion Torrent (a subsidiary of Life Technologies, Carlsbad, Calif.).
• Bead arrays are described in, e.g., U.S. Pat. Nos. 6,266,459; 6,355,431; 6,770,441; 6,859,570; 6,210,891; 6,258,568; and 6,274,320; U.S. Pat. Application Publication Nos.
• the bead array includes a plurality of beads.
• the bead array can include at least 10,000 beads (e.g., at least 100,000 beads, at least 1,000,000 beads, at least 5,000,000 beads, at least 10,000,000 beads).
• the plurality of beads includes a single type of beads (e.g., substantially uniform in size, shape, and other physical properties, such as translucence).
• the plurality of beads includes two or more types of different beads.
• a bead array is formed when beads are embedded in a hydrogel layer where the hydrogel polymerizes and secures the relative bead positions.
• the bead-arrays can be pre-equilibrated and combined with reaction buffers and enzymes (e.g., reverse-transcription mix).
• the bead arrays are frozen.
• a “flexible array” includes a plurality of spatially-barcoded features attached to, or embedded in, a flexible substrate (e.g., a membrane or tape) placed onto a biological sample.
• a flexible array includes a plurality of spatially-barcoded features embedded within a hydrogel matrix. To form such an array, features of a microarray are copied into a hydrogel, and the size of the hydrogel is reduced by removing water. These steps can be performed multiple times.
• a method for preparing a high-density spatially barcoded array can include copying a plurality of features from a microarray into a first hydrogel, where the first hydrogel is in contact with the microarray; reducing the size of the first hydrogel including the copied features by removing water, forming a first shrunken hydrogel including the copied features; copying the features in the first shrunken hydrogel into a second hydrogel, where the second hydrogel is in contact with the first hydrogel; and reducing the size of the second hydrogel including the copied features by removing water, forming a second shrunken hydrogel including the copied features, thus generating a high-density spatially barcoded array.
• the result is a high-density flexible array including spatially-barcoded features.
• spatially-barcoded beads can be loaded onto a substrate (e.g., a hydrogel) to produce a high-density self-assembled bead array.
• a substrate e.g., a hydrogel
• Flexible arrays can be pre-equilibrated, combined with reaction buffers and enzymes at functional concentrations (e.g., a reverse-transcription mix).
• the flexible bead-arrays can be stored for extended periods (e.g., days) or frozen until ready for use.
• permeabilization of biological samples e.g., a tissue section
• the flexible array can be placed directly on the sample, or placed in indirect contact with the biological sample (e.g., with an intervening layer or substance between the biological sample and the flexible bead-array).
• reverse transcription and targeted capture of analytes can be performed on solid microspheres, or circular beads of a first size and circular beads of a second size.
• a “microcapillary array” is an arrayed series of features that are partitioned by microcapillaries.
• a “microcapillary channel” is an individual partition created by the microcapillaries.
• microcapillary channels can be fluidically isolated from other microcapillary channels, such that fluid or other contents in one microcapillary channel in the array are separated from fluid or other contents in a neighboring microcapillary channel in the array.
• the density and order of the microcapillaries can be any suitable density or order of discrete sites.
• microcapillary arrays are treated to generate conditions that facilitate loading.
• An example is the use of a corona wand (BD-20AC, Electro Technic Products) to generate a hydrophilic surface.
• a feature e.g., a bead with capture probe attached
• BD-20AC Electro Technic Products
• a feature is loaded onto a microcapillary array such that the exact position of the feature within the array is known.
• a capture probe containing a spatial barcode can be placed into a microcapillary channel so that the spatial barcode can enable identification of the location from which the barcode sequence of the barcoded nucleic acid molecule was derived.
• empirical testing can be performed to generate loading/distribution conditions that facilitate a single feature per microcapillary. In some embodiments, it can be desirable to achieve distribution conditions that facilitate only a single feature (e.g., bead) per microcapillary channel. In some embodiments, it can be desirable to achieve distribution conditions that facilitate more than one feature (e.g., bead) per microcapillary channel, by flowing the features through the microcapillary channel.
• the microcapillary array is placed in contact with a sample (e.g., on top or below) so that microcapillaries containing a feature (e.g., a bead, which can include a capture probe) are in contact with the biological sample.
• a biological sample is placed onto an exposed side of a microcapillary array and mechanical compression is applied, moving the biological sample into the microcapillary channel to create a fluidically isolated reaction chamber containing the biological sample.
• a biological sample is partitioned by contacting a microcapillary array to the biological sample, thereby creating microcapillary channels including a bead and a portion of the biological sample.
• a portion of a biological sample contained in a microcapillary channel is one or more cells.
• a feature is introduced into a microcapillary array by flow after one or more cells are added to a microcapillary channel.
• reagents are added to the microcapillary array.
• the added reagents can include enzymatic reagents, and reagent mixtures for performing amplification of a nucleic acid.
• the reagents include a reverse transcriptase, a ligase, one or more nucleotides, and any combinations thereof.
• One or more microcapillary channels can be sealed after reagents are added to the microcapillary channels, e.g. using silicone oil, mineral oil, a non-porous material, or lid.
• a reagent solution is removed from each microcapillary channel following an incubation for an amount of time and at a certain temperature or range of temperatures, e.g., following a hybridization or an amplification reaction.
• Reagent solutions can be processed individually for sequencing, or pooled for sequencing analysis.
• an array can include a capture probe attached directly or indirectly to the substrate.
• the capture probe includes a capture domain (e.g., a nucleotide sequence) that can specifically bind (e.g., hybridize) to a target analyte (e.g., mRNA, DNA, or protein) within a sample.
• a target analyte e.g., mRNA, DNA, or protein
• the binding of the capture probe to the target can be detected and quantified by detection of a visual signal, e.g. a fluorophore, a heavy metal (e.g., silver ion), or chemiluminescent label, which has been incorporated into the target.
• the intensity of the visual signal correlates with the relative abundance of each analyte in the biological sample. Since an array can contain thousands or millions of capture probes (or more), an array of features with capture probes can interrogate many analytes in parallel.
• a substrate includes one or more capture probes that are designed to capture analytes from one or more organisms.
• a substrate can contain one or more capture probes designed to capture mRNA from one organism (e.g., a human) and one or more capture probes designed to capture DNA from a second organism (e.g., a bacterium).
• the capture probes can be attached to a substrate or feature using a variety of techniques.
• the capture probe is directly attached to a feature that is fixed on an array.
• the capture probes are immobilized to a substrate by chemical immobilization.
• a chemical immobilization can take place between functional groups on the substrate and corresponding functional elements on the capture probes.
• Exemplary corresponding functional elements in the capture probes can either be an inherent chemical group of the capture probe, e.g. a hydroxyl group, or a functional element can be introduced on to the capture probe.
• An example of a functional group on the substrate is an amine group.
• the capture probe to be immobilized includes a functional amine group or is chemically modified in order to include a functional amine group. Means and methods for such a chemical modification are well known in the art.
• the capture probe is a nucleic acid. In some embodiments, the capture probe is immobilized on the feature or the substrate via its 5′ end. In some embodiments, the capture probe is immobilized on a feature or a substrate via its 5′ end and includes from the 5′ to 3′ end: one or more barcodes (e.g., a spatial barcode and/or a UMI) and one or more capture domains. In some embodiments, the capture probe is immobilized on a feature via its 5′ end and includes from the 5′ to 3′ end: one barcode (e.g., a spatial barcode or a UMI) and one capture domain.
• one barcode e.g., a spatial barcode or a UMI
• the capture probe is immobilized on a feature or a substrate via its 5′ end and includes from the 5′ to 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., a spatial barcode and/or a UMI), and a capture domain.
• the capture probe is immobilized on a feature or a substrate via its 5′ end and includes from the 5′ to 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., a spatial barcode and/or a UMI), a second functional domain, and a capture domain.
• the capture probe is immobilized on a feature or a substrate via its 5′ end and includes from the 5′ to 3′ end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain.
• the capture probe is immobilized on a feature or a substrate via its 5′ end and does not include a spatial barcode.
• the capture probe is immobilized on a feature or a substrate via its 5′ end and does not include a UMI.
• the capture probe includes a sequence for initiating a sequencing reaction.
• the capture probe is immobilized on a feature or a substrate via its 3′ end. In some embodiments, the capture probe is immobilized on a feature or a substrate via its 3′ end and includes from the 3′ to 5′ end: one or more barcodes (e.g., a spatial barcode and/or a UMI) and one or more capture domains. In some embodiments, the capture probe is immobilized on a feature or a substrate via its 3′ end and includes from the 3′ to 5′ end: one barcode (e.g., a spatial barcode or a UMI) and one capture domain.
• one barcode e.g., a spatial barcode or a UMI
• the capture probe is immobilized on a feature or a substrate via its 3′ end and includes from the 3′ to 5′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., a spatial barcode and/or a UMI), and a capture domain.
• the capture probe is immobilized on a feature or a substrate via its 3′ end and includes from the 3′ to 5′ end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain.
• a capture probe can further include a substrate (e.g., a support attached to the capture probe, a support attached to the feature, or a support attached to the substrate).
• a typical substrate for a capture probe to be immobilized includes moieties which are capable of binding to such capture probes, e.g., to amine-functionalized nucleic acids. Examples of such substrates are carboxy, aldehyde, or epoxy supports.
• the substrates on which capture probes can be immobilized can be chemically activated, e.g. by the activation of functional groups, available on the substrate.
• activated substrate relates to a material in which interacting or reactive chemical functional groups are established or enabled by chemical modification procedures.
• a substrate including carboxyl groups can be activated before use.
• certain substrates contain functional groups that can react with specific moieties already present in the capture probes.
• a covalent linkage is used to directly couple a capture probe to a substrate.
• a capture probe is indirectly coupled to a substrate through a linker separating the “first” nucleotide of the capture probe from the substrate, i.e., a chemical linker.
• a capture probe does not bind directly to the array, but interacts indirectly, for example by binding to a molecule which itself binds directly or indirectly to the array.
• the capture probe is indirectly attached to a substrate (e.g., via a solution including a polymer).
• the capture probe can further include an upstream sequence (5′ to the sequence that hybridizes to the nucleic acid, e.g. RNA of the tissue sample) that is capable of hybridizing to 5′ end of the surface probe.
• the capture domain of the capture probe can be seen as a capture domain oligonucleotide, which can be used in the synthesis of the capture probe in embodiments where the capture probe is immobilized on the array indirectly.
• a substrate is comprised of an inert material or matrix (e.g., glass slides) that has been functionalization by, for example, treatment with a material comprising reactive groups which enable immobilization of capture probes.
• an inert material or matrix e.g., glass slides
• a material comprising reactive groups which enable immobilization of capture probes.
• Non-limiting examples include polyacrylamide hydrogels supported on an inert substrate (e.g., glass slide; see WO 2005/065814 and U.S. Patent Application No. 2008/0280773, the entire contents of which are incorporated herein by reference).
• functionalized biomolecules are immobilized on a functionalized substrate using covalent methods.
• Methods for covalent attachment include, for example, condensation of amines and activated carboxylic esters (e.g., N-hydroxysuccinimide esters); condensation of amine and aldehydes under reductive amination conditions; and cycloaddition reactions such as the Diels-Alder [4+2] reaction, 1,3-dipolar cycloaddition reactions, and [2+2] cycloaddition reactions.
• Methods for covalent attachment also include, for example, click chemistry reactions, including [3+2] cycloaddition reactions (e.g., Huisgen 1,3-dipolar cycloaddition reaction and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)); thiol-ene reactions; the Diels-Alder reaction and inverse electron demand Diels-Alder reaction; [4+1] cycloaddition of isonitriles and tetrazines; and nucleophilic ring-opening of small carbocycles (e.g., epoxide opening with amino oligonucleotides).
• click chemistry reactions including [3+2] cycloaddition reactions (e.g., Huisgen 1,3-dipolar cycloaddition reaction and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)); thiol-en
• Methods for covalent attachment also include, for example, maleimides and thiols; andpara-nitrophenyl ester-functionalized oligonucleotides and polylysine-functionalized substrate.
• Methods for covalent attachment also include, for example, disulfide reactions; radical reactions (see, e.g., U.S. Pat. No. 5,919,626, the entire contents of which are herein incorporated by reference); and hydrazide-functionalized substrate (e.g., wherein the hydrazide functional group is directly or indirectly attached to the substrate) and aldehyde-functionalized oligonucleotides (see, e.g., Yershov et al. (1996) Proc. Natl. Acad. Sci. USA 93, 4913-4918, the entire contents of which are herein incorporated by reference).
• functionalized biomolecules are immobilized on a functionalized substrate using photochemical covalent methods.
• Methods for photochemical covalent attachment include, for example, immobilization of antraquinone-conjugated oligonucleotides (see, e.g., Koch et al. (2000) Bioconjugate Chem. 11, 474-483, the entire contents of which are herein incorporated by reference).
• functionalized biomolecules e.g., capture probes are immobilized on a functionalized substrate using non-covalent methods.
• Methods for non-covalent attachment include, for example, biotin-functionalized oligonucleotides and streptavidin-treated substrates (see, e.g., Holmstrom et al. (1993) Analytical Biochemistry 209, 278-283 and Gilles et al. (1999) Nature Biotechnology 17, 365-370, the entire contents of which are herein incorporated by reference).
• an oligonucleotide e.g., a capture probe
• a substrate or feature according to the methods set forth in U.S. Pat. Nos. 6,737,236, 7,259,258, 7,375,234, 7,427,678, 5,610,287, 5,807,522, 5,837,860, and 5,472,881; U.S. Patent Application Publication Nos. 2008/0280773 and 2011/0059865; Shalon et al. (1996) Genome Research, 639-645; Rogers et al. (1999) Analytical Biochemistry 266, 23-30; Stimpson et al. (1995) Proc. Natl. Acad. Sci.
• arrays can be prepared by a variety of methods.
• arrays are prepared through the synthesis (e.g., in-situ synthesis) of oligonucleotides on the array, or by jet printing or lithography.
• synthesis e.g., in-situ synthesis
• light-directed synthesis of high-density DNA oligonucleotides can be achieved by photolithography or solid-phase DNA synthesis.
• synthetic linkers modified with photochemical protecting groups can be attached to a substrate and the photochemical protecting groups can be modified using a photolithographic mask (applied to specific areas of the substrate) and light, thereby producing an array having localized photo-deprotection.
• the arrays are “spotted” or “printed” with oligonucleotides and these oligonucleotides (e.g., capture probes) are then attached to the substrate.
• the oligonucleotides can be applied by either noncontact or contact printing.
• a noncontact printer can use the same method as computer printers (e.g., bubble jet or inkjet) to expel small droplets of probe solution onto the substrate.
• the specialized inkjet-like printer can expel nanoliter to picoliter volume droplets of oligonucleotide solution, instead of ink, onto the substrate.
• each print pin directly applies the oligonucleotide solution onto a specific location on the surface.
• the oligonucleotides can be attached to the substrate surface by the electrostatic interaction of the negative charge of the phosphate backbone of the DNA with a positively charged coating of the substrate surface or by UV-cross-linked covalent bonds between the thymidine bases in the DNA and amine groups on the treated substrate surface.
• the substrate is a glass slide.
• the oligonucleotides e.g., capture probes
• the substrate is a covalent bond to a chemical matrix, e.g. epoxy-silane, amino-silane, lysine, polyacrylamide, etc.
• the arrays can also be prepared by in situ-synthesis. In some embodiments, these arrays can be prepared using photolithography. The method typically relies on UV masking and light-directed combinatorial chemical synthesis on a substrate to selectively synthesize probes directly on the surface of the array, one nucleotide at a time per spot, for many spots simultaneously.
• a substrate contains covalent linker molecules that have a protecting group on the free end that can be removed by light. UV light is directed through a photolithographic mask to deprotect and activate selected sites with hydroxyl groups that initiate coupling with incoming protected nucleotides that attach to the activated sites.
• the mask is designed in such a way that the exposure sites can be selected, and thus specify the coordinates on the array where each nucleotide can be attached.
• the process can be repeated, a new mask is applied activating different sets of sites and coupling different bases, allowing arbitrary oligonucleotides to be constructed at each site.
• This process can be used to synthesize hundreds of thousands of different oligonucleotides.
• maskless array synthesizer technology can be used. It uses an array of programmable micromirrors to create digital masks that reflect the desired pattern of UV light to deprotect the features.
• the inkjet spotting process can also be used for in-situ oligonucleotide synthesis.
• the different nucleotide precursors plus catalyst can be printed on the substrate, and are then combined with coupling and deprotection steps. This method relies on printing picoliter volumes of nucleotides on the array surface in repeated rounds of base-by-base printing that extends the length of the oligonucleotide probes on the array.
• Arrays can also be prepared by active hybridization via electric fields to control nucleic acid transport. Negatively charged nucleic acids can be transported to specific sites, or features, when a positive current is applied to one or more test sites on the array.
• the surface of the array can contain a binding molecule, e.g., streptavidin, which allows for the formation of bonds (e.g., streptavidin-biotin bonds) once electronically addressed biotinylated probes reach their targeted location.
• bonds e.g., streptavidin-biotin bonds
• An array for spatial analysis can be generated by various methods as described herein.
• the array has a plurality of capture probes comprising spatial barcodes. These spatial barcodes and their relationship to the locations on the array can be determined. In some cases, such information is readily available, because the oligonucleotides are spotted, printed, or synthesized on the array with a pre-determined pattern.
• the spatial barcode can be decoded by methods described herein, e.g., by in-situ sequencing, by various labels associated with the spatial barcodes etc.
• an array can be used as a template to generate a daughter array. Thus, the spatial barcode can be transferred to the daughter array with a known pattern.
• an array comprising barcoded probes can be generated through ligation of a plurality of oligonucleotides.
• an oligonucleotide of the plurality contains a portion of a barcode, and the complete barcode is generated upon ligation of the plurality of oligonucleotides.
• a first oligonucleotide containing a first portion of a barcode can be attached to a substrate (e.g., using any of the methods of attaching an oligonucleotide to a substrate described herein), and a second oligonucleotide containing a second portion of the barcode can then be ligated onto the first oligonucleotide to generate a complete barcode.
• a substrate e.g., using any of the methods of attaching an oligonucleotide to a substrate described herein
• a second oligonucleotide containing a second portion of the barcode can then be ligated onto the first oligonucleotide to generate a complete barcode.
• Different combinations of the first, second and any additional portions of a barcode can be used to increase the diversity of the barcodes.
• the first and/or the second oligonucleotide can be attached to the substrate via a surface linker which contains a cleavage site.
• the ligated oligonucleotide is linearized by cleaving at the cleavage site.
• a plurality of second oligonucleotides comprising two or more different barcode sequences can be ligated onto a plurality of first oligonucleotides that comprise the same barcode sequence, thereby generating two or more different species of barcodes.
• a first oligonucleotide attached to a substrate containing a first portion of a barcode can initially be protected with a protective group (e.g., a photocleavable protective group), and the protective group can be removed prior to ligation between the first and second oligonucleotide.
• a protective group e.g., a photocleavable protective group
• a concentration gradient of the oligonucleotides can be applied to a substrate such that different combinations of the oligonucleotides are incorporated into a barcoded probe depending on its location on the substrate.
• Barcoded probes on an array can also be generated by adding single nucleotides to existing oligonucleotides on an array, for example, using polymerases that function in a template-independent manner.
• Single nucleotides can be added to existing oligonucleotides in a concentration gradient, thereby generating probes with varying length, depending on the location of the probes on the array.
• Arrays can also be prepared by modifying existing arrays, for example, by modifying the oligonucleotides attached to the arrays.
• probes can be generated on an array that comprises oligonucleotides that are attached to the array at the 3′ end and have a free 5′ end.
• the oligonucleotides can be in situ synthesized oligonucleotides, and can include a barcode.
• the length of the oligonucleotides can be less than 50 nucleotides (nts) (e.g., less than 45, 40, 35, 30, 25, 20, 15, or 10 nts).
• a primer complementary to a portion of an oligonucleotide can be used to hybridize with the oligonucleotide and extend (using the oligonucleotide as a template) to form a duplex and to create a 3′ overhang.
• the 3′ overhang thus allows additional nucleotides or oligonucleotides to be added on to the duplex.
• a capture probe can be generated by, for instance, adding one or more oligonucleotides to the end of the 3′ overhang (e.g., via splint oligonucleotide mediated ligation), where the added oligonucleotides can include the sequence or a portion of the sequence of a capture domain.
• oligonucleotides on an existing array include a recognition sequence that can hybridize with a splint oligonucleotide
• probes can also be generated by directly ligating additional oligonucleotides onto the existing oligonucleotides via the splint oligonucleotide.
• the recognition sequence can at the free 5′ end or the free 3′ end of an oligonucleotide on the existing array.
• Recognition sequences useful for the methods of the present disclosure may not contain restriction enzyme recognition sites or secondary structures (e.g., hairpins), and may include high contents of Guanine and Cytosine nucleotides and thus have high stability.
• Bead arrays can be generated by attaching beads (e.g., barcoded beads) to a substrate in a regular pattern, or an irregular arrangement. Beads can be attached to selective regions on a substrate by, e.g., selectively activating regions on the substrate to allow for attachment of the beads. Activating selective regions on the substrate can include activating a coating (e.g., a photocleavable coating) or a polymer that is applied on the substrate. Beads can be attached iteratively, e.g., a subset of the beads can be attached at one time, and the same process can be repeated to attach the remaining beads. Alternatively, beads can be attached to the substrate all in one step.
• beads e.g., barcoded beads
• Barcoded beads, or beads comprising a plurality of barcoded probes can be generated by first preparing a plurality of barcoded probes on a substrate, depositing a plurality of beads on the substrate, and generating probes attached to the beads using the probes on the substrate as a template.
• oligonucleotides Large scale commercial manufacturing methods allow for millions of oligonucleotides to be attached to an array.
• Commercially available arrays include those from Roche NimbleGen, Inc., (Wisconsin) and Affymetrix (ThermoFisher Scientific).
• arrays can be prepared according to the methods set forth in WO 2012/140224, WO 2014/060483, WO 2016/162309, WO 2017/019456, WO 2018/091676, and WO 2012/140224, and U.S. Patent Application No. 2018/0245142.
• the entire contents of the foregoing documents are herein incorporated by reference.
• a feature on the array includes a bead.
• two or more beads are dispersed onto a substrate to create an array, where each bead is a feature on the array. Beads can optionally be dispersed into wells on a substrate, e.g., such that only a single bead is accommodated per well.
• a “bead” is a particle.
• a bead can be porous, non-porous, solid, semi-solid, and/or a combination thereof.
• a bead can be dissolvable, disruptable, and/or degradable, whereas in certain embodiments, a bead is not degradable.
• a bead can generally be of any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, oval, oblong, amorphous, circular, cylindrical, and variations thereof.
• a cross section (e.g., a first cross-section) can correspond to a diameter or maximum cross-sectional dimension of the bead. In some embodiments, the bead can be approximately spherical. In such embodiments, the first cross-section can correspond to the diameter of the bead. In some embodiments, the bead can be approximately cylindrical. In such embodiments, the first cross-section can correspond to a diameter, length, or width along the approximately cylindrical bead.
• Beads can be of uniform size or heterogeneous size. “Polydispersity” generally refers to heterogeneity of sizes of molecules or particles.
• beads can be provided as a population or plurality of beads having a relatively monodisperse size distribution. Where it can be desirable to provide relatively consistent amounts of reagents, maintaining relatively consistent bead characteristics, such as size, can contribute to the overall consistency.
• the beads provided herein can have size distributions that have a coefficient of variation in their cross-sectional dimensions of less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, or lower.
• a plurality of beads provided herein has a polydispersity index of less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or lower.
• the bead can have a diameter or maximum dimension no larger than 100 ⁇ m (e.g., no larger than 95 ⁇ m, 90 ⁇ m, 85 ⁇ m, 80 ⁇ m, 75 ⁇ m, 70 ⁇ m, 65 ⁇ m, 60 ⁇ m, 55 ⁇ m, 50 ⁇ m, 45 ⁇ m, 40 ⁇ m, 35 ⁇ m, 30 ⁇ m, 25 ⁇ m, 20 ⁇ m, 15 ⁇ m, 14 ⁇ m, 13 ⁇ m, 12 ⁇ m, 11 ⁇ m, m, 9 ⁇ m, 8 ⁇ m, 7 ⁇ m, 6 ⁇ m, 5 ⁇ m, 4 ⁇ m, 3 ⁇ m, 2 ⁇ m, or 1 ⁇ m.)
• a plurality of beads has an average diameter no larger than 100 ⁇ m.
• a plurality of beads has an average diameter or maximum dimension no larger than 95 ⁇ m, 90 ⁇ m, 85 ⁇ m, 80 ⁇ m, 75 ⁇ m, 70 ⁇ m, 65 ⁇ m, 60 ⁇ m, 55 ⁇ m, 50 ⁇ m, 45 ⁇ m, 40 ⁇ m, 35 ⁇ m, 30 ⁇ m, 25 ⁇ m, 20 ⁇ m, 15 ⁇ m, 14 ⁇ m, 13 ⁇ m, 12 ⁇ m, 11 ⁇ m, 10 ⁇ m, 9 ⁇ m, 8 ⁇ m, 7 ⁇ m, 6 ⁇ m, 5 ⁇ m, 4 ⁇ m, 3 ⁇ m, 2 ⁇ m, or 1 ⁇ m.
• the volume of the bead can be at least about 1 ⁇ m 3 , e.g., at least 1 ⁇ m 3 , 2 ⁇ m 3 , 3 ⁇ m 3 , 4 ⁇ m 3 , 5 ⁇ m 3 , 6 ⁇ m 3 , 7 ⁇ m 3 , 8 ⁇ m 3 , 9 ⁇ m 3 , 10 ⁇ m 3 , 12 ⁇ m 3 , 14 m 3 , 16 ⁇ m 3 , 18 ⁇ m 3 , 20 ⁇ m 3 , 25 ⁇ m 3 , 30 ⁇ m 3 , 35 ⁇ m 3 , 40 ⁇ m 3 , 45 ⁇ m 3 , 50 ⁇ m 3 , 55 ⁇ m 3 , 60 ⁇ m 3 , 65 ⁇ m 3 , 70 ⁇ m 3 , 75 ⁇ m 3 , 80 ⁇ m 3 , 85 ⁇ m 3 , 90 ⁇ m 3 , 95 ⁇ m 3 , 100 ⁇ m 3 , 125 ⁇ m 3 ,
• the bead can have a volume of between about 1 ⁇ m 3 and 100 ⁇ m 3 , such as between about 1 ⁇ m 3 and 10 ⁇ m 3 , between about 10 ⁇ m 3 and 50 ⁇ m 3 , or between about 50 ⁇ m 3 and 100 ⁇ m 3 .
• the bead can include a volume of between about 100 ⁇ m 3 and 1000 ⁇ m 3 , such as between about 100 ⁇ m 3 and 500 ⁇ m 3 or between about 500 ⁇ m 3 and 1000 ⁇ m 3 .
• the bead can include a volume between about 1000 ⁇ m 3 and 3000 ⁇ m 3 , such as between about 1000 ⁇ m 3 and 2000 ⁇ m 3 or between about 2000 ⁇ m 3 and 3000 ⁇ m 3 . In some embodiments, the bead can include a volume between about 1 ⁇ m 3 and 3000 ⁇ m 3 , such as between about 1 ⁇ m 3 and 2000 ⁇ m 3 , between about 1 ⁇ m 3 and 1000 ⁇ m 3 , between about 1 ⁇ m 3 and 500 ⁇ m 3 , or between about 1 ⁇ m 3 and 250 ⁇ m 3 .
• the bead can include one or more cross-sections that can be the same or different.
• the bead can have a first cross-section that is different from a second cross-section.
• the bead can have a first cross-section that is at least about 0.0001 micrometer, 0.001 micrometer, 0.01 micrometer, 0.1 micrometer, or 1 micrometer.
• the bead can include a cross-section (e.g., a first cross-section) of at least about 1 micrometer (m), 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, 8 ⁇ m, 9 ⁇ m, 10 ⁇ m, 11 ⁇ m, 12 ⁇ m, 13 ⁇ m, 14 ⁇ m, 15 ⁇ m, 16 am, 17 ⁇ m, 18 ⁇ m, 19 ⁇ m, 20 ⁇ m, 25 ⁇ m, 30 ⁇ m, 35 ⁇ m, 40 ⁇ m, 45 ⁇ m, 50 ⁇ m, 55 ⁇ m, 60 ⁇ m, 65 ⁇ m, 70 ⁇ m, 75 ⁇ m, 80 ⁇ m, 85 ⁇ m, 90 ⁇ m, 100 ⁇ m, 120 ⁇ m, 140 ⁇ m, 160 ⁇ m, 180 ⁇ m, 200 am, 250 ⁇ m, 300 ⁇ m, 350 ⁇ m, 400 ⁇ m, 450 ⁇ m
• the bead can include a cross-section (e.g., a first cross-section) of between about 1 ⁇ m and 500 ⁇ m, such as between about 1 ⁇ m and 100 ⁇ m, between about 100 ⁇ m and 200 ⁇ m, between about 200 m and 300 ⁇ m, between about 300 ⁇ m and 400 ⁇ m, or between about 400 ⁇ m and 500 ⁇ m.
• the bead can include a cross-section (e.g., a first cross-section) of between about 1 m and 100 ⁇ m.
• the bead can have a second cross-section that is at least about 1 ⁇ m.
• the bead can include a second cross-section of at least about 1 micrometer (am), 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, 8 ⁇ m, 9 ⁇ m, 10 ⁇ m, 11 ⁇ m, 12 ⁇ m, 13 ⁇ m, 14 ⁇ m, 15 ⁇ m, 16 ⁇ m, 17 ⁇ m, 18 ⁇ m, 19 ⁇ m, 20 ⁇ m, 25 ⁇ m, 30 ⁇ m, 35 ⁇ m, 40 ⁇ m, 45 ⁇ m, 50 ⁇ m, 55 ⁇ m, 60 ⁇ m, 65 ⁇ m, 70 ⁇ m, 75 ⁇ m, 80 ⁇ m, 85 ⁇ m, 90 ⁇ m, 100 ⁇ m, 120 ⁇ m, 140 ⁇ m, 160 ⁇ m, 180 ⁇ m, 200 ⁇ m, 250 ⁇ m, 300 ⁇ m, 350 ⁇ m, 400 ⁇ m, 450 ⁇ m, 500 ⁇ m, 550 ⁇
• the bead can include a second cross-section of between about 1 ⁇ m and 500 ⁇ m, such as between about 1 ⁇ m and 100 ⁇ m, between about 100 ⁇ m and 200 ⁇ m, between about 200 ⁇ m and 300 ⁇ m, between about 300 ⁇ m and 400 ⁇ m, or between about 400 ⁇ m and 500 ⁇ m.
• the bead can include a second cross-section of between about 1 ⁇ m and 100 ⁇ m.
• beads can be of a nanometer scale (e.g., beads can have a diameter or maximum cross-sectional dimension of about 100 nanometers (nm) to about 900 nanometers (nm) (e.g., 850 nm or less, 800 nm or less, 750 nm or less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less).
• nm nanometer scale
• a plurality of beads can have an average diameter or average maximum cross-sectional dimension of about 100 nanometers (nm) to about 900 nanometers (nm) (e.g., 850 nm or less, 800 nm or less, 750 nm or less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less).
• a bead has a diameter or size that is about the size of a single cell (e.g., a single cell under evaluation).
• the bead can be a gel bead.
• a “gel” is a semi-rigid material permeable to liquids and gases.
• Exemplary gels include, but are not limited to, those having a colloidal structure, such as agarose; polymer mesh structures, such as gelatin; hydrogels; and cross-linked polymer structures, such as polyacrylamide, SFA (see, for example, U.S. Patent Application Publication No. 2011/0059865, which is incorporated herein by reference in its entirety) and PAZAM (see, for example, U.S. Patent Application Publication No. 2014/0079923, which is incorporated herein by reference in its entirety).
• a gel can be formulated into various shapes and dimensions depending on the context of intended use.
• a gel is prepared and formulated as a gel bead (e.g., a gel bead including capture probes attached or associated with the gel bead).
• a gel bead can be a hydrogel bead.
• a hydrogel bead can be formed from molecular precursors, such as a polymeric or monomeric species.
• a hydrogel bead can include a polymer matrix (e.g., a matrix formed by polymerization or cross-linking).
• a polymer matrix can include one or more polymers (e.g., polymers having different functional groups or repeat units).
• Cross-linking can be via covalent, ionic, and/or inductive interactions, and/or physical entanglement.
• a semi-solid bead can be a liposomal bead.
• Solid beads can include metals including, without limitation, iron oxide, gold, and silver.
• the bead can be a silica bead.
• the bead can be rigid.
• the bead can be flexible and/or compressible.
• the bead can be a macromolecule.
• the bead can be formed of nucleic acid molecules bound together.
• the bead can be formed via covalent or non-covalent assembly of molecules (e.g., macromolecules), such as monomers or polymers.
• Polymers or monomers can be natural or synthetic.
• Polymers or monomers can be or include, for example, nucleic acid molecules (e.g., DNA or RNA).
• a bead can be rigid, or flexible and/or compressible.
• a bead can include a coating including one or more polymers. Such a coating can be disruptable or dissolvable.
• a bead includes a spectral or optical label (e.g., dye) attached directly or indirectly (e.g., through a linker) to the bead.
• a bead can be prepared as a colored preparation (e.g., a bead exhibiting a distinct color within the visible spectrum) that can change color (e.g., colorimetric beads) upon application of a desired stimulus (e.g., heat and/or chemical reaction) to form differently colored beads (e.g., opaque and/or clear beads).
• a bead can include natural and/or synthetic materials.
• a bead can include a natural polymer, a synthetic polymer or both natural and synthetic polymers.
• natural polymers include, without limitation, proteins, sugars such as deoxyribonucleic acid, rubber, cellulose, starch (e.g., amylose, amylopectin), enzymes, polysaccharides, silks, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum, corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate, or natural polymers thereof.
• Examples of synthetic polymers include, without limitation, acrylics, nylons, silicones, spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethanes, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethylene terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(oxymethylene), polyformaldehyde, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene dichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and/or combinations (e.g., co-poly
• a bead is a degradable bead.
• a degradable bead can include one or more species (e.g., disulfide linkers, primers, other oligonucleotides, etc.) with a labile bond such that, when the bead/species is exposed to the appropriate stimuli, the labile bond is broken and the bead degrades.
• the labile bond can be a chemical bond (e.g., covalent bond, ionic bond) or can be another type of physical interaction (e.g., van der Waals interactions, dipole-dipole interactions, etc.).
• a crosslinker used to generate a bead can include a labile bond.
• the labile bond can be broken and the bead degraded.
• the disulfide bonds of the cystamine can be broken and the bead degraded.
• Degradation can refer to the disassociation of a bound or entrained species (e.g., disulfide linkers, primers, other oligonucleotides, etc.) from a bead, both with and without structurally degrading the physical bead itself.
• entrained species can be released from beads through osmotic pressure differences due to, for example, changing chemical environments.
• alteration of bead pore sizes due to osmotic pressure differences can generally occur without structural degradation of the bead itself.
• an increase in pore size due to osmotic swelling of a bead can permit the release of entrained species within the bead.
• osmotic shrinking of a bead can cause a bead to better retain an entrained species due to pore size contraction.
• any suitable agent that can degrade beads can be used.
• changes in temperature or pH can be used to degrade thermo-sensitive or pH-sensitive bonds within beads.
• chemical degrading agents can be used to degrade chemical bonds within beads by oxidation, reduction or other chemical changes.
• a chemical degrading agent can be a reducing agent, such as DTT, where DTT can degrade the disulfide bonds formed between a crosslinker and gel precursors, thus degrading the bead.
• a reducing agent can be added to degrade the bead, which can cause the bead to release its contents.
• Examples of reducing agents can include, without limitation, dithiothreitol (DTT), (3-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof.
• DTT dithiothreitol
• DTBA (2S)-2-amino-1,4-dimercaptobutane
• TCEP tris(2-carboxyethyl) phosphine
• any of a variety of chemical agents can be used to trigger the degradation of beads.
• chemical agents include, but are not limited to, pH-mediated changes to the integrity of a component within the bead, degradation of a component of a bead via cleavage of cross-linked bonds, and depolymerization of a component of a bead.
• a bead can be formed from materials that include degradable chemical crosslinkers, such as N,N′-bis-(acryloyl)cystamine (BAC) or cystamine. Degradation of such degradable crosslinkers can be accomplished through any variety of mechanisms.
• a bead can be contacted with a chemical degrading agent that can induce oxidation, reduction or other chemical changes.
• a chemical degrading agent can be a reducing agent, such as dithiothreitol (DTT).
• reducing agents can include ⁇ -mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof.
• aqueous solution such as water
• hydrolytic degradation can trigger hydrolytic degradation, and thus degradation of the bead.
• Beads can also be induced to release their contents upon the application of a thermal stimulus.
• a change in temperature can cause a variety of changes to a bead.
• heat can cause a solid bead to liquefy.
• a change in heat can cause melting of a bead such that a portion of the bead degrades.
• heat can increase the internal pressure of the bead components such that the bead ruptures or explodes. Heat can also act upon heat-sensitive polymers used as materials to construct beads.
• degradable beads it can be beneficial to avoid exposing such beads to the stimulus or stimuli that cause such degradation prior to a given time, in order to, for example, avoid premature bead degradation and issues that arise from such degradation, including for example poor flow characteristics and aggregation.
• beads include reducible cross-linking groups, such as disulfide groups
• reducing agents e.g., DTT or other disulfide cleaving reagents.
• treatment of the beads described herein will, in some embodiments be provided free of reducing agents, such as DTT.
• reducing agent free (or DTT free) enzyme preparations in treating the beads described herein.
• enzymes include, e.g., polymerase enzyme preparations, reverse transcriptase enzyme preparations, ligase enzyme preparations, as well as many other enzyme preparations that can be used to treat the beads described herein.
• the terms “reducing agent free” or “DTT free” preparations refer to a preparation having less than about 1/10th, less than about 1/50th, or less than about 1/100th of the lower ranges for such materials used in degrading the beads.
• the reducing agent free preparation can have less than about 0.01 millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or less than about 0.0001 mM DTT.
• the amount of DTT can be undetectable.
• a degradable bead can be useful to more quickly release an attached capture probe (e.g., a nucleic acid molecule, a spatial barcode sequence, and/or a primer) from the bead when the appropriate stimulus is applied to the bead as compared to a bead that does not degrade.
• an attached capture probe e.g., a nucleic acid molecule, a spatial barcode sequence, and/or a primer
• the species can have greater mobility and accessibility to other species in solution upon degradation of the bead.
• a species can also be attached to a degradable bead via a degradable linker (e.g., disulfide linker).
• the degradable linker can respond to the same stimuli as the degradable bead or the two degradable species can respond to different stimuli.
• a capture probe having one or more spatial barcodes can be attached, via a disulfide bond, to a polyacrylamide bead including cystamine.
• the bead Upon exposure of the spatially barcoded bead to a reducing agent, the bead degrades and the capture probe having the one or more spatial barcode sequences is released upon breakage of both the disulfide linkage between the capture probe and the bead and the disulfide linkages of the cystamine in the bead.
• Each type of labile bond can be sensitive to an associated stimulus (e.g., chemical stimulus, light, temperature, pH, enzymes, etc.) such that release of reagents attached to a bead via each labile bond can be controlled by the application of the appropriate stimulus.
• an associated stimulus e.g., chemical stimulus, light, temperature, pH, enzymes, etc.
• ester linkage e.g., clea
• a bond can be cleavable via other nucleic acid molecule targeting enzymes, such as restriction enzymes (e.g., restriction endonucleases). Such functionality can be useful in controlled release of reagents from a bead.
• another reagent including a labile bond can be linked to a bead after gel bead formation via, for example, an activated functional group of the bead as described above.
• a gel bead including a labile bond is reversible.
• a gel bead with a reversible labile bond is used to capture one or more regions of interest of a biological sample.
• a bead including a thermolabile bond can be heated by a light source (e.g., a laser) that causes a change in the gel bead that facilitates capture of a biological sample in contact with the gel bead.
• Capture probes having one or more spatial barcodes that are releasably, cleavably, or reversibly attached to the beads described herein include capture probes that are released or releasable through cleavage of a linkage between the capture probe and the bead, or that are released through degradation of the underlying bead itself, allowing the capture probes having the one or more spatial barcodes to be accessed or become accessible by other reagents, or both.
• Beads can have different physical properties. Physical properties of beads can be used to characterize the beads. Non-limiting examples of physical properties of beads that can differ include size, shape, circularity, density, symmetry, and hardness. For example, beads can be of different sizes. Different sizes of beads can be obtained by using microfluidic channel networks configured to provide specific sized beads (e.g., based on channel sizes, flow rates, etc.). In some embodiments, beads have different hardness values that can be obtained by varying the concentration of polymer used to generate the beads. In some embodiments, a spatial barcode attached to a bead can be made optically detectable using a physical property of the capture probe.
• a nucleic acid origami such as a deoxyribonucleic acid (DNA) origami
• DNA deoxyribonucleic acid
• a nucleic acid molecule, or a plurality of nucleic acid molecules can be folded to create two- and/or three-dimensional geometric shapes. The different geometric shapes can be optically detected.
• nanoparticles with more than one distinct physical property can be used to make the beads physically distinguishable.
• Janus particles with both hydrophilic and hydrophobic surfaces can be used to provide unique physical properties.
• a bead is able to identify multiple analytes (e.g., nucleic acids, proteins, chromatin, metabolites, drugs, gRNA, and lipids) from a single cell.
• a bead is able to identify a single analyte from a single cell (e.g., mRNA).
• a bead can have a tunable pore size.
• the pore size can be chosen to, for instance, retain denatured nucleic acids.
• the pore size can be chosen to maintain diffusive permeability to exogenous chemicals such as sodium hydroxide (NaOH) and/or endogenous chemicals such as inhibitors.
• a bead can be formed of a biocompatible and/or biochemically compatible material, and/or a material that maintains or enhances cell viability.
• a bead can be formed from a material that can be depolymerized thermally, chemically, enzymatically, and/or optically.
• beads can be non-covalently loaded with one or more reagents.
• the beads can be non-covalently loaded by, for instance, subjecting the beads to conditions sufficient to swell the beads, allowing sufficient time for the reagents to diffuse into the interiors of the beads, and subjecting the beads to conditions sufficient to de-swell the beads.
• Swelling of the beads can be accomplished, for instance, by placing the beads in a thermodynamically favorable solvent, subjecting the beads to a higher or lower temperature, subjecting the beads to a higher or lower ion concentration, and/or subjecting the beads to an electric field.
• the swelling of the beads can be accomplished by various swelling methods.
• swelling is reversible (e.g., by subjecting beads to conditions that promote de-swelling).
• the de-swelling of the beads is accomplished, for instance, by transferring the beads in a thermodynamically unfavorable solvent, subjecting the beads to lower or higher temperatures, subjecting the beads to a lower or higher ion concentration, and/or adding or removing an electric field.
• the de-swelling of the beads can be accomplished by various de-swelling methods.
• de-swelling is reversible (e.g., subject beads to conditions that promote swelling).
• the de-swelling of beads can include transferring the beads to cause pores in the bead to shrink.
• the shrinking can then hinder reagents within the beads from diffusing out of the interiors of the beads.
• the hindrance created can be due to steric interactions between the reagents and the interiors of the beads.
• the transfer can be accomplished microfluidically. For instance, the transfer can be achieved by moving the beads from one co-flowing solvent stream to a different co-flowing solvent stream.
• the swellability and/or pore size of the beads can be adjusted by changing the polymer composition of the bead.
• a bead can include a polymer that is responsive to temperature so that when the bead is heated or cooled, the characteristics or dimensions of the bead can change.
• a polymer can include poly(N-isopropylacrylamide).
• a gel bead can include poly(N-isopropylacrylamide) and when heated the gel bead can decrease in one or more dimensions (e.g., a cross-sectional diameter, multiple cross-sectional diameters).
• a temperature sufficient for changing one or more characteristics of the gel bead can be, for example, at least about 0 degrees Celsius (° C.), 1° C., 2° C., 3° C., 4° C., 5° C., 10° C., or higher.
• the temperature can be about 4° C.
• a temperature sufficient for changing one or more characteristics of the gel bead can be, for example, at least about 25° C., 30° C., 35° C., 37° C., 40° C., 45° C., 50° C., or higher.
• the temperature can be about 37° C.
• Functionalization of beads for attachment of capture probes can be achieved through a wide range of different approaches, including, without limitation, activation of chemical groups within a polymer, incorporation of active or activatable functional groups in the polymer structure, or attachment at the pre-polymer or monomer stage in bead production.
• the bead can be functionalized to bind to targeted analytes, such as nucleic acids, proteins, carbohydrates, lipids, metabolites, peptides, or other analytes.
• a bead can contain molecular precursors (e.g., monomers or polymers), which can form a polymer network via polymerization of the molecular precursors.
• a precursor can be an already polymerized species capable of undergoing further polymerization via, for example, a chemical cross-linkage.
• a precursor can include one or more of an acrylamide or a methacrylamide monomer, oligomer, or polymer.
• the bead can include prepolymers, which are oligomers capable of further polymerization. For example, polyurethane beads can be prepared using prepolymers.
• a bead can contain individual polymers that can be further polymerized together (e.g., to form a co-polymer).
• a bead can be generated via polymerization of different precursors, such that they include mixed polymers, co-polymers, and/or block co-polymers.
• a bead can include covalent or ionic bonds between polymeric precursors (e.g., monomers, oligomers, and linear polymers), nucleic acid molecules (e.g., oligonucleotides), primers, and other entities.
• covalent bonds can be carbon-carbon bonds or thioether bonds.
• Cross-linking of polymers can be permanent or reversible, depending upon the particular cross-linker used. Reversible cross-linking can allow the polymer to linearize or dissociate under appropriate conditions. In some embodiments, reversible cross-linking can also allow for reversible attachment of a material bound to the surface of a bead. In some embodiments, a cross-linker can form a disulfide linkage. In some embodiments, a chemical cross-linker forming a disulfide linkage can be cystamine or a modified cystamine.
• the activation agent can include a cross-linking agent, or a chemical that activates a cross-linking agent within formed droplets.
• the activation agent can include a polymerization initiator.
• the polymer precursor includes a mixture of acrylamide monomer with a N,N′-bis-(acryloyl)cystamine (BAC) comonomer
• an agent such as tetraethylmethylenediamine (TEMED) can be provided, which can initiate the copolymerization of the acrylamide and BAC into a cross-linked polymer network, or other conditions sufficient to polymerize or gel the precursors.
• the conditions sufficient to polymerize or gel the precursors can include exposure to heating, cooling, electromagnetic radiation, and/or light.
• a polymer or gel can be formed.
• the polymer or gel can be diffusively permeable to chemical or biochemical reagents.
• the polymer or gel can be diffusively impermeable to macromolecular constituents.
• the polymer or gel can include one or more of disulfide cross-linked polyacrylamide, agarose, alginate, polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, or elastin.
• the polymer or gel can include any other polymer or gel.
• disulfide linkages can be formed between molecular precursor units (e.g., monomers, oligomers, or linear polymers) or precursors incorporated into a bead and nucleic acid molecules (e.g., oligonucleotides, capture probes).
• Cystamine is an organic agent including a disulfide bond that can be used as a crosslinker agent between individual monomeric or polymeric precursors of a bead.
• Polyacrylamide can be polymerized in the presence of cystamine or a species including cystamine (e.g., a modified cystamine) to generate polyacrylamide gel beads including disulfide linkages (e.g., chemically degradable beads including chemically-reducible cross-linkers).
• the disulfide linkages can permit the bead to be degraded (or dissolved) upon exposure of the bead to a reducing agent.
• chitosan a linear polysaccharide polymer
• cross-linked with glutaraldehyde via hydrophilic chains can be achieved by chemical reactions that are initiated by heat, pressure, change in pH, and/or radiation.
• a bead can include an acrydite moiety, which in certain aspects can be used to attach one or more capture probes to the bead.
• an acrydite moiety can refer to an acrydite analogue generated from the reaction of acrydite with one or more species (e.g., disulfide linkers, primers, other oligonucleotides, etc.), such as, without limitation, the reaction of acrydite with other monomers and cross-linkers during a polymerization reaction.
• species e.g., disulfide linkers, primers, other oligonucleotides, etc.
• Acrydite moieties can be modified to form chemical bonds with a species to be attached, such as a capture probe.
• Acrydite moieties can be modified with thiol groups capable of forming a disulfide bond or can be modified with groups already including a disulfide bond.
• the thiol or disulfide (via disulfide exchange) can be used as an anchor point for a species to be attached or another part of the acrydite moiety can be used for attachment.
• attachment can be reversible, such that when the disulfide bond is broken (e.g., in the presence of a reducing agent), the attached species is released from the bead.
• an acrydite moiety can include a reactive hydroxyl group that can be used for attachment of species.
• precursors that are polymerized to form a bead can include acrydite moieties, such that when a bead is generated, the bead also includes acrydite moieties.
• the acrydite moieties can be attached to a nucleic acid molecule (e.g., an oligonucleotide), which can include a priming sequence (e.g., a primer for amplifying target nucleic acids, random primer, primer sequence for messenger RNA) and/or one or more capture probes.
• the one or more capture probes can include sequences that are the same for all capture probes coupled to a given bead and/or sequences that are different across all capture probes coupled to the given bead.
• the capture probe can be incorporated into the bead. In some embodiments, the capture probe can be incorporated or attached to the bead such that the capture probe retains a free 3′ end. In some embodiments, the capture probe can be incorporated or attached to the bead such that the capture probe retains a free 5′ end. In some embodiments, beads can be functionalized such that each bead contains a plurality of different capture probes.
• a bead can include a plurality of capture probes e.g., Capture Probe 1, Capture Probe 2, and Capture Probe 3, and each of Capture Probes 1, Capture Probes 2, and Capture Probes 3 contain a distinct capture domain (e.g., capture domain of Capture Probe 1 includes a poly(dT) capture domain, capture domain of Capture Probe 2 includes a gene-specific capture domain, and capture domain of Capture Probe 3 includes a CRISPR-specific capture domain).
• capture domain of Capture Probe 1 includes a poly(dT) capture domain
• capture domain of Capture Probe 2 includes a gene-specific capture domain
• capture domain of Capture Probe 3 includes a CRISPR-specific capture domain
• precursors that are polymerized to form a bead can include a functional group that is reactive or capable of being activated such that when it becomes reactive it can be polymerized with other precursors to generate beads including the activated or activatable functional group.
• the functional group can then be used to attach additional species (e.g., disulfide linkers, primers, other oligonucleotides, etc.) to the beads.
• additional species e.g., disulfide linkers, primers, other oligonucleotides, etc.
• some precursors including a carboxylic acid (COOH) group can co-polymerize with other precursors to form a bead that also includes a COOH functional group.
• acrylic acid (a species including free COOH groups), acrylamide, and bis(acryloyl)cystamine can be co-polymerized together to generate a bead including free COOH groups.
• the COOH groups of the bead can be activated (e.g., via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) or 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)) such that they are reactive (e.g., reactive to amine functional groups where EDC/NHS or DMTMM are used for activation).
• EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
• NHS N-Hydroxysuccinimide
• DTMM 4-(4,6-Dimethoxy-1,3,5-triazin
• the activated COOH groups can then react with an appropriate species (e.g., a species including an amine functional group where the carboxylic acid groups are activated to be reactive with an amine functional group) as a functional group on a moiety to be linked to the bead.
• an appropriate species e.g., a species including an amine functional group where the carboxylic acid groups are activated to be reactive with an amine functional group
• Beads including disulfide linkages in their polymeric network can be functionalized with additional species (e.g., disulfide linkers, primers, other oligonucleotides, etc.) via reduction of some of the disulfide linkages to free thiols.
• the disulfide linkages can be reduced via, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.) to generate free thiol groups, without dissolution of the bead.
• a reducing agent e.g., DTT, TCEP, etc.
• Free thiols of the beads can then react with free thiols of a species or a species including another disulfide bond (e.g., via thiol-disulfide exchange) such that the species can be linked to the beads (e.g., via a generated disulfide bond).
• free thiols of the beads can react with any other suitable group.
• free thiols of the beads can react with species including an acrydite moiety.
• the free thiol groups of the beads can react with the acrydite via Michael addition chemistry, such that the species including the acrydite is linked to the bead.
• uncontrolled reactions can be prevented by inclusion of a thiol capping agent such as N-ethylmalieamide or iodoacetate.
• Activation of disulfide linkages within a bead can be controlled such that only a small number of disulfide linkages are activated. Control can be exerted, for example, by controlling the concentration of a reducing agent used to generate free thiol groups and/or concentration of reagents used to form disulfide bonds in bead polymerization.
• a low concentration of reducing agent e.g., molecules of reducing agent:gel bead ratios
• reducing agent e.g., molecules of reducing agent:gel bead ratios
• reducing agent e.g., molecules of reducing agent:gel bead ratios
• controlling the number of disulfide linkages that are reduced to free thiols can be useful in ensuring bead structural integrity during functionalization.
• optically-active agents such as fluorescent dyes can be coupled to beads via free thiol groups of the beads and used to quantify the number of free thiols present in a bead and/or track a bead.
• addition of moieties to a bead after bead formation can be advantageous.
• addition of a capture probe after bead formation can avoid loss of the species (e.g., disulfide linkers, primers, other oligonucleotides, etc.) during chain transfer termination that can occur during polymerization.
• species e.g., disulfide linkers, primers, other oligonucleotides, etc.
• smaller precursors e.g., monomers or cross linkers that do not include side chain groups and linked moieties
• functionalization after bead synthesis can minimize exposure of species (e.g., oligonucleotides) to be loaded with potentially damaging agents (e.g., free radicals) and/or chemical environments.
• the generated hydrogel can possess an upper critical solution temperature (UCST) that can permit temperature driven swelling and collapse of a bead.
• UST upper critical solution temperature
• Such functionality can aid in oligonucleotide (e.g., a primer) infiltration into the bead during subsequent functionalization of the bead with the oligonucleotide.
• Post-production functionalization can also be useful in controlling loading ratios of species in beads, such that, for example, the variability in loading ratio is minimized.
• Species loading can also be performed in a batch process such that a plurality of beads can be functionalized with the species in a single batch.
• Reagents can be encapsulated in beads during bead generation (e.g., during polymerization of precursors). Such reagents can or cannot participate in polymerization. Such reagents can be entered into polymerization reaction mixtures such that generated beads include the reagents upon bead formation. In some embodiments, such reagents can be added to the beads after formation.
• Such reagents can include, for example, capture probes (e.g., oligonucleotides), reagents for a nucleic acid amplification reaction (e.g., primers, polymerases, dNTPs, co-factors (e.g., ionic co-factors), buffers) including those described herein, reagents for enzymatic reactions (e.g., enzymes, co-factors, substrates, buffers), reagents for nucleic acid modification reactions such as polymerization, ligation, or digestion, and/or reagents for template preparation (e.g., tagmentation) for one or more sequencing platforms (e.g., Nextera® (e.g., transposase-based sequencing) for Illumina® (next-generation sequencing system)).
• capture probes e.g., oligonucleotides
• reagents for a nucleic acid amplification reaction e.g., primers, poly
• Such reagents can include one or more enzymes described herein, including without limitation, polymerase, reverse transcriptase, restriction enzymes (e.g., endonuclease), transposase, ligase, proteinase K, DNAse, etc.
• Such reagents can also or alternatively include one or more reagents such as lysis agents, inhibitors, inactivating agents, chelating agents, stimulus agents. Trapping of such reagents can be controlled by the polymer network density generated during polymerization of precursors, control of ionic charge within the bead (e.g., via ionic species linked to polymerized species), or by the release of other species.
• Encapsulated reagents can be released from a bead upon bead degradation and/or by application of a stimulus capable of releasing the reagents from the bead.
• the beads can also include (e.g., encapsulate or have attached thereto) a plurality of capture probes that include spatial barcodes, and the optical properties of the spatial barcodes can be used for optical detection of the beads.
• the absorbance of light by the spatial barcodes can be used to distinguish the beads from one another.
• a detectable label can directly or indirectly attach to a spatial barcode and provide optical detection of the bead.
• each bead in a group of one or more beads has a unique detectable label, and detection of the unique detectable label determines the location of the spatial barcode sequence associated with the bead.
• Optical properties giving rise to optical detection of beads can be due to optical properties of the bead surface (e.g., a detectable label attached to the bead or the size of the bead), or optical properties from the bulk region of the bead (e.g., a detectable label incorporated during bead formation or an optical property of the bead itself).
• a detectable label can be associated with a bead or one or more moieties coupled to the bead.
• the beads include a plurality of detectable labels.
• a fluorescent dye can be attached to the surface of the beads and/or can be incorporated into the beads.
• Different intensities of the different fluorescent dyes can be used to increase the number of optical combinations that can be used to differentiate between beads. For example, if N is the number of fluorescent dyes (e.g., between 2 and 10 fluorescent dyes, such as 4 fluorescent dyes) and M is the possible intensities for the dyes (e.g., between 2 and 50 intensities, such as 20 intensities), then M N are the possible distinct optical combinations. In one example, 4 fluorescent dyes with 20 possible intensities can be used to generate 160,000 distinct optical combinations.
• One or more optical properties of the beads or biological contents can be used to distinguish the individual beads or biological contents from other beads or biological contents.
• the beads are made optically detectable by including a detectable label having optical properties to distinguish the beads from one another.
• optical properties of the beads can be used for optical detection of the beads.
• optical properties can include absorbance, birefringence, color, fluorescence, luminosity, photosensitivity, reflectivity, refractive index, scattering, or transmittance.
• beads can have different birefringence values based on degree of polymerization, chain length, or monomer chemistry.
• nanobeads such as quantum dots or Janus beads
• a quantum dot can be attached to a spatial barcode of a bead.
• Optical labels of beads can provide enhanced spectral resolution to distinguish between beads with unique spatial barcodes (e.g., beads including unique spatial barcode sequences).
• a first bead includes a first optical label and spatial barcodes each having a first spatial barcode sequence.
• a second bead includes a second optical label and spatial barcodes each having a second spatial barcode sequence.
• the first optical label and second optical label can be different (e.g., provided by two different fluorescent dyes or the same fluorescent dye at two different intensities).
• the first and second spatial barcode sequences can be different nucleic acid sequences.
• the beads can be imaged to identify the first and second optical labels, and the first and second optical barcodes can then be used to associate the first and second optical labels with the first and second spatial barcode sequences, respectively.
• Optical labels can be included while generating the beads.
• optical labels can be included in the polymer structure of a gel bead, or attached at the pre-polymer or monomer stage in bead production.
• the beads include moieties that attach to one or more optical labels (e.g., at a surface of a bead and/or within a bead).
• optical labels can be loaded into the beads with one or more reagents.
• reagents and optical labels can be loaded into the beads by diffusion of the reagents (e.g., a solution of reagents including the optical barcodes).
• optical labels can be included while preparing spatial barcodes.
• spatial barcodes can be prepared by synthesizing molecules including barcode sequences (e.g., using a split pool or combinatorial approach).
• Optical labels can be attached to spatial barcodes prior to attaching the spatial barcodes to a bead.
• optical labels can be included after attaching spatial barcodes to a bead.
• optical labels can be attached to spatial barcodes coupled to the bead.
• spatial barcodes or sequences thereof can be releasably or cleavably attached to the bead.
• Optical labels can be releasably or non-releasably attached to the bead.
• a first bead (e.g., a bead including a plurality of spatial barcodes) can be coupled to a second bead including one or more optical labels.
• the first bead can be covalently coupled to the second bead via a chemical bond.
• the first bead can be non-covalently associated with the second bead.
• the first and/or second bead can include a plurality of spatial barcodes.
• the plurality of spatial barcodes coupled to a given bead can include the same barcode sequences.
• the first and second beads can include spatial barcodes including the same barcode sequences or different barcode sequences.
• Bead arrays containing captured analytes can be processed in bulk or partitioned into droplet emulsions for preparing sequencing libraries.
• next generation sequencing reads are clustered and correlated to the spatial position of the spatial barcode on the bead array.
• the information can be computationally superimposed over a high-resolution image of the tissue section to identify the location(s), where the analytes were detected.
• de-cross linking can be performed to account for de-crosslinking chemistries that may be incompatible with certain barcoding/library prep biochemistry (e.g., presence of proteases).
• a two-step process is possible. In the first step, beads can be provided in droplets such that DNA binds to the beads after the conventional de-crosslinking chemistry is performed. In the second step, the emulsion is broken and beads collected and then re-encapsulated after washing for further processing.
• beads can be affixed or attached to a substrate using photochemical methods.
• a bead can be functionalized with perfluorophenylazide silane (PFPA silane), contacted with a substrate, and then exposed to irradiation (see, e.g., Liu et al. (2006) Journal of the American Chemical Society 128, 14067-14072).
• PFPA silane perfluorophenylazide silane
• immobilization of antraquinone-functionalized substrates see, e.g., Koch et al. (2000) Bioconjugate Chem. 11, 474-483, the entire contents of which are herein incorporated by reference).
• the arrays can also be prepared by bead self-assembly.
• Each bead can be covered with hundreds of thousands of copies of a specific oligonucleotide. In some embodiments, each bead can be covered with about 1,000 to about 1,000,000 oligonucleotides. In some embodiments, each bead can be covered with about 1,000,000 to about 10,000,000 oligonucleotides. In some embodiments, each bead can covered with about 2,000,000 to about 3,000,000, about 3,000,000 to about 4,000,000, about 4,000,000 to about 5,000,000, about 5,000,000 to about 6,000,000, about 6,000,000 to about 7,000,000, about 7,000,000 to about 8,000,000, about 8,000,000 to about 9,000,000, or about 9,000,000 to about 10,000,000 oligonucleotides.
• each bead can be covered with about 10,000,000 to about 100,000,000 oligonucleotides. In some embodiments, each bead can be covered with about 100,000,000 to about 1,000,000,000 oligonucleotides. In some embodiments, each bead can be covered with about 1,000,000,000 to about 10,000,000,000 oligonucleotides.
• the beads can be irregularly distributed across etched substrates during the array production process. During this process, the beads can be self-assembled into arrays (e.g., on a fiber-optic bundle substrate or a silica slide substrate). In some embodiments, the beads irregularly arrive at their final location on the array. Thus, the bead location may need to be mapped or the oligonucleotides may need to be synthesized based on a predetermined pattern.
• Beads can be affixed or attached to a substrate covalently, non-covalently, with adhesive, or a combination thereof.
• the attached beads can be, for example, layered in a monolayer, a bilayer, a trilayer, or as a cluster.
• a “monolayer” generally refers to an arrayed series of probes, beads, spots, dots, features, micro-locations, or islands that are affixed or attached to a substrate, such that the beads are arranged as one layer of single beads. In some embodiments, the beads are closely packed.
• the phrase “substantial monolayer” or “substantially form(s) a monolayer” generally refers to (the formation of) an arrayed series of probes, beads, microspheres, spots, dots, features, micro-locations, or islands that are affixed or attached to a substrate, such that about 50% to about 99% (e.g., about 50% to about 98%) of the beads are arranged as one layer of single beads. This arrangement can be determined using a variety of methods, including microscopic imaging.
• the monolayer of beads is a located in a predefined area on the substrate.
• the predefined area can be partitioned with physical barriers, a photomask, divots in the substrate, or wells in the substrate.
• reactive element generally refers to a molecule or molecular moiety that can react with another molecule or molecular moiety to form a covalent bond.
• Reactive elements include, for example, amines, aldehydes, alkynes, azides, thiols, haloacetyls, pyridyl disulfides, hydrazides, carboxylic acids, alkoxyamines, sulfhydryls, maleimides, Michael acceptors, hydroxyls, and active esters.
• Some reactive elements can be treated with one or more activating agents (e.g., acylating agents, isourea-forming agents) to increase susceptibility of the reactive element to nucleophilic attack.
• activating agents include N-hydroxysuccinimide, N-hydroxysulfosuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, dicyclohexylcarbodiimide, diisopropylcarbodiiimide, 1-hydroxybenzotriazole, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexfluorophosphate, (benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, 4-(N,N-dimethylamino)pyridine, and carbonyldiimidazole.
• the reactive element is bound directly to a bead.
• hydrogel beads can be treated with an acrylic acid monomer to form acrylic acid-functionalized hydrogel beads.
• the reactive element is bound indirectly to the bead via one or more linkers.
• a “linker” generally refers to a multifunctional (e.g., bifunctional, trifunctional) reagent used for conjugating two or more chemical moieties.
• a linker can be a cleavable linker that can undergo induced dissociation.
• the dissociation can be induced by a solvent (e.g., hydrolysis and solvolysis); by irradiation (e.g., photolysis); by an enzyme (e.g., enzymolysis); or by treatment with a solution of specific pH (e.g., pH 4, 5, 6, 7, or 8).
• a solvent e.g., hydrolysis and solvolysis
• irradiation e.g., photolysis
• an enzyme e.g., enzymolysis
• a solution of specific pH e.g., pH 4, 5, 6, 7, or 8
• the reactive element is bound directly to a substrate.
• a glass slide can be coated with (3-aminopropyl)triethoxysilane.
• the reactive element is bound indirectly to a substrate via one or more linkers.
• beads e.g., optically labeled beads, hydrogel beads, microsphere beads
• the beads are coupled to a substrate via a covalent bond between a first reactive element and a second reactive element.
• the covalently-bound beads substantially form a monolayer of beads (e.g., hydrogel beads, microsphere beads) on the substrate.
• the beads are functionalized with a first reactive element, which is directly bound to the beads. In some embodiments, the beads are functionalized with a first reactive element, which is indirectly bound to the beads via a linker.
• the linker is a benzophenone. In some embodiments, the linker is an amino methacrylamide. For example, the linker can be 3-aminopropyl methacrylamide. In some embodiments, the linker is a PEG linker. In some embodiments, the linker is a cleavable linker.
• the substrate is functionalized with a second reactive element, which is directly bound to the substrate. In some embodiments, the substrate is functionalized with a second reactive element, which is indirectly bound to the beads via a linker.
• the linker is a benzophenone.
• the linker can be benzophenone.
• the linker is an amino methacrylamide.
• the linker can be 3-aminopropyl methacrylamide.
• the linker is a PEG linker. In some embodiments, the linker is a cleavable linker.
• the substrate is a glass slide. In some embodiments, the substrate is a pre-functionalized glass slide.
• about 99% of the covalently-bound beads form a monolayer of beads on the substrate.
• about 50% to about 98% form a monolayer of beads on the substrate.
• about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, or about 50% to about 55% of the covalently-bound beads form a monolayer of beads on the substrate.
• about 55% to about 98%, about 60% to about 98%, about 65% to about 98%, about 70% to about 98%, about 75% to about 98%, about 80% to about 98%, about 85% to about 98%, about 90% to about 95%, or about 95% to about 98% of the covalently-bound beads form a monolayer of beads on the substrate.
• about 55% to about 95%, about 60% to about 90%, about 65% to about 95%, about 70% to about 95%, about 75% to about 90%, about 75% to about 95%, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, or about 85% to about 95% of the covalently-bound beads for a monolayer of beads on the substrate.
• At least one of the first reactive element and the second reactive element is selected from the group consisting of:
• R 1 is selected from H, C 1 -C 6 alkyl, or —SO 3 ;
• R 2 is C 1 -C 6 alkyl
• X is a halo moiety
• At least one of the first reactive element or the second reactive element comprises
• the bead e.g., hydrogel bead or microsphere bead
• At least one of the first reactive element or the second reactive element is selected from the group consisting of:
• R 1 is selected from H, C 1 -C 6 alkyl, or —SO 3 ;
• R 2 is C 1 -C 6 alkyl
• X is a halo moiety
• At least one of the first reactive element or the second reactive element comprises
• R 1 is selected from H, C 1 -C 6 alkyl, or —SO 3 .
• R 1 is H.
• R 1 is C 1 -C 6 alkyl.
• R 1 is —SO 3 .
• At least one of the first reactive element or the second reactive element comprises
• R 2 is C 1 -C 6 alkyl. In some embodiments, R 2 is methyl.
• At least one of the first reactive element or the second reactive element comprises
• the active ester is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
• the activating agent is an acylating agent (e.g., N-hydroxysuccinimide and N-hydroxysulfosuccinimide).
• the activating agent is an O-acylisourea-forming agent (e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide, and diisopropylcarbodiiimide).
• the activating agent is a combination of at least one acylating agent and at least one O-isourea-forming agents (e.g., N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysulfosuccinimide (sulfo-NHS), and a combination thereof).
• O-isourea-forming agents e.g., N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysulfosuccinimide (sulfo-NHS), and a combination thereof.
• At least one of the first reactive element or the second reactive element comprises
• At least one of the first reactive element or the second reactive element comprises
• X is a halo moiety.
• X is chloro, bromo, or iodo.
• At least one of the first reactive element or the second reactive element comprises
• At least one of the first reactive element or the second reactive element comprises
• At least one of the first reactive element or the second reactive element comprises
• At least one of the first reactive element or the second reactive element is selected from the group consisting of:
• R 3 is H or C 1 -C 6 alkyl
• R 4 is H or trimethylsilyl.
• At least one of the first reactive element or the second reactive element comprises
• R 4 is H or trimethylsilyl. In some embodiments, R 4 is H.
• At least one of the first reactive element or the second reactive element is selected from the group consisting of:
• R 3 is H or C 1 -C 6 alkyl. In some embodiments, R 3 is H. In some embodiments, R 3 is C 1 -C 6 alkyl.
• At least one of the first reactive element or the second reactive element comprises
• R 3 is H or C 1 -C 6 alkyl. In some embodiments, R 3 is H. In some embodiments, R 3 is C 1 -C 6 alkyl.
• At least one of the first reactive elements or the second reactive elements comprises
• At least one of the first reactive elements or the second reactive elements comprises
• one of the first reactive elements or the second reactive elements is selected from the group consisting of:
• R 1 is selected from H, C 1 -C 6 alkyl, or —SO 3 ;
• R 2 is C 1 -C 6 alkyl
• X is a halo moiety
• first reactive element or the second reactive element is selected from the group consisting of:
• R 3 is H or C 1 -C 6 alkyl
• R 4 is H or trimethylsilyl.
• one of the first reactive elements or the second reactive elements is selected from the group consisting of
• R 3 is H or C 1 -C 6 alkyl; and the other of the first reactive element or the second reactive element is
• R 4 is H or trimethylsilyl.
• R 3 is H.
• R 3 is C 1 -C 6 alkyl.
• R 4 is H.
• R 4 is trimethylsilyl.
• one of the first reactive element or the second reactive element is selected from the group consisting of:
• R 1 is selected from H, C 1 -C 6 alkyl, or —SO 3 ;
• R 2 is C 1 -C 6 alkyl
• X is a halo moiety
• first reactive element or the second reactive element is selected from the group consisting of:
• R 3 is H or C 1 -C 6 alkyl.
• R 1 is H.
• R 1 is C 1 -C 6 alkyl.
• R 1 is —SO 3 .
• R 2 is methyl.
• X is iodo.
• R 3 is H.
• R 3 is C 1 -C 6 alkyl.
• one of the first reactive elements or the second reactive elements is selected from the group consisting of:
• R 1 is selected from H, C 1 -C 6 alkyl, or —SO 3 ;
• R 2 is C 1 -C 6 alkyl
• R 3 is H or C 1 -C 6 alkyl.
• R 1 is H.
• R 1 is C 1 -C 6 alkyl.
• R 1 is —SO 3 .
• R 2 is methyl.
• R 3 is H.
• R 3 is C 1 -C 6 alkyl.
• one of the first reactive element or the second reactive element is selected from the group consisting of:
• X is a halo moiety
• X is bromo. In some embodiments, X is iodo.
• one of the first reactive element or the second reactive element is selected from the group consisting of
• halo refers to fluoro (F), chloro (Cl), bromo (Br), or iodo (I).
• alkyl refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms.
• C 1-10 indicates that the group may have from 1 to 10 (inclusive) carbon atoms in it.
• Non-limiting examples include methyl, ethyl, iso-propyl, tert-butyl, n-hexyl.
• haloalkyl refers to an alkyl, in which one or more hydrogen atoms is/are replaced with an independently selected halo.
• alkoxy refers to an —O-alkyl radical (e.g., —OCH 3 ).
• alkylene refers to a divalent alkyl (e.g., —CH 2 —).
• alkenyl refers to a hydrocarbon chain that may be a straight chain or branched chain having one or more carbon-carbon double bonds.
• the alkenyl moiety contains the indicated number of carbon atoms. For example, C 2-6 indicates that the group may have from 2 to 6 (inclusive) carbon atoms in it.
• alkynyl refers to a hydrocarbon chain that may be a straight chain or branched chain having one or more carbon-carbon triple bonds.
• the alkynyl moiety contains the indicated number of carbon atoms. For example, C 2-6 indicates that the group may have from 2 to 6 (inclusive) carbon atoms in it.
• aryl refers to a 6-20 carbon mono-, bi-, tri- or polycyclic group wherein at least one ring in the system is aromatic (e.g., 6-carbon monocyclic, 10-carbon bicyclic, or 14-carbon tricyclic aromatic ring system); and wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent.
• aryl groups include phenyl, naphthyl, tetrahydronaphthyl, and the like.
• beads e.g., optically-labeled beads, hydrogel beads, or microsphere beads
• beads are coupled to a substrate via a non-covalent bond between a first affinity group and a second affinity group.
• the non-covalently-bound beads substantially form a monolayer of beads (e.g., hydrogel beads, microsphere beads) on the substrate.
• the beads are functionalized with a first affinity group, which is directly bound to the beads. In some embodiments, the beads are functionalized with a first affinity group, which is indirectly bound to the beads via a linker.
• the linker is a benzophenone. In some embodiments, the linker is an amino methacrylamide. For example, the linker can be 3-aminopropyl methacrylamide. In some embodiments, the linker is a PEG linker. In some embodiments, the linker is a cleavable linker.
• the substrate is functionalized with a second affinity group, which is directly bound to the substrate. In some embodiments, the substrate is functionalized with a second affinity group, which is indirectly bound to the beads via a linker.
• the linker is a benzophenone. In some embodiments, the linker is an amino methacrylamide. For example, the linker can be 3-aminopropyl methacrylamide. In some embodiments, the linker is a PEG linker. In some embodiments, the linker is a cleavable linker.
• first affinity group or the second affinity group is biotin, and the other of the first affinity group or the second affinity group is streptavidin.
• about 99% of the non-covalently-bound beads form a monolayer of beads on the substrate.
• about 50% to about 98% form a monolayer of beads on the substrate.
• about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, or about 50% to about 55% of the non-covalently-bound beads form a monolayer of beads on the substrate.
• about 55% to about 98%, about 60% to about 98%, about 65% to about 98%, about 70% to about 98%, about 75% to about 98%, about 80% to about 98%, about 85% to about 98%, about 90% to about 95%, or about 95% to about 98% of the non-covalently-bound beads form a monolayer of beads on the substrate.
• the monolayer of beads is a formed in a predefined area on the substrate.
• the predefined area is partitioned with physical barriers. For example, divots or wells in the substrate.
• the predefined area is partitioned using a photomask.
• the substrate is coated with a photo-activated solution, dried, then irradiated under a photomask.
• the photo-activated solution is UV-activated.
• an “adhesive” generally refers to a substance used for sticking objects or materials together.
• Adhesives include, for example, glues, pastes, liquid tapes, epoxy, bioadhesives, gels, and mucilage.
• an adhesive is liquid tape.
• the adhesive is glue.
• beads are adhered to a substrate using an adhesive (e.g., liquid tape, glue, paste).
• the adhered beads substantially form a monolayer of beads on the substrate (e.g., a glass slide).
• the beads are hydrogel beads.
• the beads are microsphere beads.
• the beads are coated with the adhesive, and then the beads are contacted with the substrate.
• the substrate is coated with the adhesive, and then the substrate is contacted with the beads.
• both the substrate is coated with the adhesive and the beads are coated with the adhesive, and then the beads and substrate are contacted with one another.
• about 99% of the adhered beads form a monolayer of beads on the substrate.
• about 50% to about 98% form a monolayer of beads on the substrate.
• about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, or about 50% to about 55% of the adhered beads form a monolayer of beads on the substrate.
• about 55% to about 98%, about 60% to about 98%, about 65% to about 98%, about 70% to about 98%, about 75% to about 98%, about 80% to about 98%, about 85% to about 98%, about 90% to about 95%, or about 95% to about 98% of the adhered beads form a monolayer of beads on the substrate.
• beads can be deposited onto a biological sample such that the deposited beads form a monolayer of beads on the biological sample (e.g., over or under the biological sample).
• beads deposited on the substrate can self-assemble into a monolayer of beads that saturate the intended surface area of the biological sample under investigation.
• bead arrays can be designed, formulated, and prepared to evaluate a plurality of analytes from a biological sample of any size or dimension.
• the concentration or density of beads (e.g., gel beads) applied to the biological sample is such that the area as a whole, or one or more regions of interest in the biological sample, is saturated with a monolayer of beads.
• the beads are contacted with the biological sample by pouring, pipetting, spraying, and the like, onto the biological sample. Any suitable form of bead deposition can be used.
• the biological sample can be confined to a specific region or area of the array.
• a biological sample can be affixed to a glass slide and a chamber, gasket, or cage positioned over the biological sample to act as a containment region or frame within which the beads are deposited.
• the density or concentration of beads needed to saturate an area or biological sample can be readily determined by one of ordinary skill in the art (e.g., through microscopic visualization of the beads on the biological sample).
• the bead array contains microfluidic channels to direct reagents to the spots or beads of the array.
• a feature of an array can have a diameter or maximum dimension between 1 ⁇ m to 100 ⁇ m. For example, between 1 ⁇ m to 10 ⁇ m, 1 ⁇ m to 20 ⁇ m, 1 ⁇ m to 30 ⁇ m, 1 ⁇ m to 40 ⁇ m, 1 ⁇ m to 50 ⁇ m, 1 ⁇ m to 60 ⁇ m, 1 ⁇ m to 70 ⁇ m, 1 ⁇ m to 80 ⁇ m, 1 ⁇ m to 90 ⁇ m, 90 ⁇ m to 100 ⁇ m, 80 ⁇ m to 100 ⁇ m, 70 ⁇ m to 100 ⁇ m, 60 ⁇ m to 100 ⁇ m, 50 ⁇ m to 100 ⁇ m, 40 ⁇ m to 100 ⁇ m, 30 ⁇ m to 100 ⁇ m, 20 m to 100 ⁇ m, or 10 ⁇ m to 100 ⁇ m.
• the feature has a diameter or maximum dimension between 30 ⁇ m to 100 ⁇ m, 40 ⁇ m to 90 ⁇ m, 50 ⁇ m to 80 ⁇ m, 60 ⁇ m to 70 ⁇ m, or any range within the disclosed sub-ranges.
• the feature has a diameter or maximum dimension no larger than 95 ⁇ m, 90 ⁇ m, 85 ⁇ m, 80 ⁇ m, 75 ⁇ m, 70 ⁇ m, 65 ⁇ m, 60 ⁇ m, 55 ⁇ m, 50 ⁇ m, 45 ⁇ m, 40 ⁇ m, 35 ⁇ m, 30 ⁇ m, 25 ⁇ m, 20 ⁇ m, 15 ⁇ m, 14 ⁇ m, 13 ⁇ m, 12 ⁇ m, 11 ⁇ m, 10 ⁇ m, 9 ⁇ m, 8 ⁇ m, 7 ⁇ m, 6 ⁇ m, 5 ⁇ m, 4 ⁇ m, 3 ⁇ m, 2 ⁇ m, or 1 ⁇ m. In some embodiments, the feature has a diameter or maximum dimension of approximately 65 ⁇ m.
• the size and/or shape of a plurality of features of an array are approximately uniform. In some embodiments, the size and/or shape of a plurality of features of an array is not uniform.
• features in an array can have an average cross-sectional dimension, and a distribution of cross-sectional dimensions among the features can have a full-width and half-maximum value of 0% or more (e.g., 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 70% or more, or 100% or more) of the average cross-sectional dimension for the distribution.
• features in an array can have an average cross-sectional dimension of between about 1 ⁇ m and about 10 ⁇ m. This range in average feature cross-sectional dimension corresponds to the approximate diameter of a single mammalian cell. Thus, an array of such features can be used to detect analytes at, or below, mammalian single-cell resolution.
• a plurality of features has a mean diameter or mean maximum dimension of about 0.1 ⁇ m to about 100 ⁇ m (e.g., about 0.1 ⁇ m to about 5 ⁇ m, about 1 ⁇ m to about 10 ⁇ m, about 1 ⁇ m to about 20 ⁇ m, about 1 ⁇ m to about 30 ⁇ m, about 1 ⁇ m to about 40 ⁇ m, about 1 ⁇ m to about 50 ⁇ m, about 1 ⁇ m to about 60 ⁇ m, about 1 ⁇ m to about 70 ⁇ m, about 1 ⁇ m to about 80 ⁇ m, about 1 ⁇ m to about 90 ⁇ m, about 90 ⁇ m to about 100 ⁇ m, about 80 ⁇ m to about 100 ⁇ m, about 70 ⁇ m to about 100 ⁇ m, about 60 ⁇ m to about 100 ⁇ m, about 50 ⁇ m to about 100 ⁇ m, about 40 ⁇ m to about 100 ⁇ m, about 30 ⁇ m to about 100 ⁇ m, about 20 ⁇ m to about 100 ⁇ m, about 1
• the plurality of features has a mean diameter or mean maximum dimension between 30 ⁇ m to 100 ⁇ m, 40 ⁇ m to 90 ⁇ m, 50 ⁇ m to 80 ⁇ m, 60 ⁇ m to 70 ⁇ m, or any range within the disclosed sub-ranges.
• the plurality of features has a mean diameter or a mean maximum dimension no larger than 95 ⁇ m, 90 ⁇ m, 85 ⁇ m, 80 ⁇ m, 75 ⁇ m, 70 ⁇ m, 65 ⁇ m, 60 ⁇ m, 55 ⁇ m, 50 ⁇ m, 45 ⁇ m, 40 ⁇ m, 35 ⁇ m, 30 ⁇ m, 25 ⁇ m, 20 ⁇ m, 15 ⁇ m, 14 ⁇ m, 13 ⁇ m, 12 ⁇ m, 11 ⁇ m, 10 ⁇ m, 9 ⁇ m, 8 ⁇ m, 7 ⁇ m, 6 ⁇ m, 5 ⁇ m, 4 ⁇ m, 3 ⁇ m, 2 ⁇ m, or 1 ⁇ m.
• the plurality of features has a mean average diameter or a mean maximum dimension of approximately 65 ⁇ m.
• the bead can have a diameter or maximum dimension no larger than 100 ⁇ m (e.g., no larger than 95 ⁇ m, 90 ⁇ m, 85 ⁇ m, 80 ⁇ m, 75 ⁇ m, 70 ⁇ m, 65 ⁇ m, 60 ⁇ m, 55 ⁇ m, 50 ⁇ m, 45 ⁇ m, 40 ⁇ m, 35 ⁇ m, 30 ⁇ m, 25 ⁇ m, 20 ⁇ m, 15 ⁇ m, 14 ⁇ m, 13 ⁇ m, 12 ⁇ m, 11 ⁇ m, 10 ⁇ m, 9 ⁇ m, 8 ⁇ m, 7 ⁇ m, 6 ⁇ m, 5 ⁇ m, 4 ⁇ m, 3 ⁇ m, 2 ⁇ m, or 1 ⁇ m).
• 100 ⁇ m e.g., no larger than 95 ⁇ m, 90 ⁇ m, 85 ⁇ m, 80 ⁇ m, 75 ⁇ m, 70 ⁇ m, 65 ⁇ m, 60 ⁇ m, 55 ⁇ m, 50 ⁇ m, 45 ⁇ m,
• a plurality of beads has an average diameter no larger than 100 ⁇ m. In some embodiments, a plurality of beads has an average diameter or maximum dimension no larger than 95 ⁇ m, 90 ⁇ m, 85 ⁇ m, 80 ⁇ m, 75 ⁇ m, 70 ⁇ m, 65 ⁇ m, 60 ⁇ m, 55 ⁇ m, 50 ⁇ m, 45 am, 40 ⁇ m, 35 ⁇ m, 30 ⁇ m, 25 ⁇ m, 20 ⁇ m, 15 ⁇ m, 14 ⁇ m, 13 ⁇ m, 12 ⁇ m, 11 ⁇ m, 10 ⁇ m, 9 ⁇ m, 8 ⁇ m, 7 ⁇ m, 6 ⁇ m, 5 ⁇ m, 4 ⁇ m, 3 ⁇ m, 2 ⁇ m, or 1 ⁇ m.
• the volume of the bead can be at least about 1 ⁇ m 3 , e.g., at least 1 ⁇ m 3 , 2 ⁇ m 3 , 3 ⁇ m 3 , 4 ⁇ m 3 , 5 ⁇ m 3 , 6 ⁇ m 3 , 7 ⁇ m 3 , 8 ⁇ m 3 , 9 ⁇ m 3 , 10 ⁇ m 3 , 12 ⁇ m 3 , 14 m 3 , 16 ⁇ m 3 , 18 ⁇ m 3 , 20 ⁇ m 3 , 25 ⁇ m 3 , 30 ⁇ m 3 , 35 ⁇ m 3 , 40 ⁇ m 3 , 45 ⁇ m 3 , 50 ⁇ m 3 , 55 ⁇ m 3 , 60 ⁇ m 3 , 65 ⁇ m 3 , 70 ⁇ m 3 , 75 ⁇ m 3 , 80 ⁇ m 3 , 85 ⁇ m 3 , 90 ⁇ m 3 , 95 ⁇ m 3 , 100 ⁇ m 3 , 125 ⁇ m 3 ,
• the bead can have a volume of between about 1 ⁇ m 3 and 100 ⁇ m 3 , such as between about 1 ⁇ m 3 and 10 ⁇ m 3 , between about 10 ⁇ m 3 and 50 ⁇ m 3 , or between about 50 ⁇ m 3 and 100 ⁇ m 3 .
• the bead can include a volume of between about 100 ⁇ m 3 and 1000 ⁇ m 3 , such as between about 100 ⁇ m 3 and 500 ⁇ m 3 or between about 500 ⁇ m 3 and 1000 ⁇ m 3 .
• the bead can include a volume between about 1000 ⁇ m 3 and 3000 ⁇ m 3 , such as between about 1000 ⁇ m 3 and 2000 ⁇ m 3 or between about 2000 ⁇ m 3 and 3000 ⁇ m 3 . In some embodiments, the bead can include a volume between about 1 ⁇ m 3 and 3000 ⁇ m 3 , such as between about 1 ⁇ m 3 and 2000 ⁇ m 3 , between about 1 ⁇ m 3 and 1000 ⁇ m 3 , between about 1 ⁇ m 3 and 500 ⁇ m 3 , or between about 1 ⁇ m 3 and 250 ⁇ m 3 .
• the bead can include one or more cross-sections that can be the same or different.
• the bead can have a first cross-section that is different from a second cross-section.
• the bead can have a first cross-section that is at least about 0.0001 micrometer, 0.001 micrometer, 0.01 micrometer, 0.1 micrometer, or 1 micrometer.
• the bead can include a cross-section (e.g., a first cross-section) of at least about 1 micrometer (m), 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, 8 ⁇ m, 9 ⁇ m, 10 ⁇ m, 11 ⁇ m, 12 ⁇ m, 13 ⁇ m, 14 ⁇ m, 15 ⁇ m, 16 am, 17 ⁇ m, 18 ⁇ m, 19 ⁇ m, 20 ⁇ m, 25 ⁇ m, 30 ⁇ m, 35 ⁇ m, 40 ⁇ m, 45 ⁇ m, 50 ⁇ m, 55 ⁇ m, 60 ⁇ m, 65 ⁇ m, 70 ⁇ m, 75 ⁇ m, 80 ⁇ m, 85 ⁇ m, 90 ⁇ m, 100 ⁇ m, 120 ⁇ m, 140 ⁇ m, 160 ⁇ m, 180 ⁇ m, 200 am, 250 ⁇ m, 300 ⁇ m, 350 ⁇ m, 400 ⁇ m, 450 ⁇ m
• the bead can include a cross-section (e.g., a first cross-section) of between about 1 ⁇ m and 500 ⁇ m, such as between about 1 ⁇ m and 100 ⁇ m, between about 100 ⁇ m and 200 ⁇ m, between about 200 m and 300 ⁇ m, between about 300 ⁇ m and 400 ⁇ m, or between about 400 ⁇ m and 500 ⁇ m.
• the bead can include a cross-section (e.g., a first cross-section) of between about 1 m and 100 ⁇ m.
• the bead can have a second cross-section that is at least about 1 ⁇ m.
• the bead can include a second cross-section of at least about 1 micrometer (am), 2 ⁇ m, 3 ⁇ m, 4 ⁇ m, 5 ⁇ m, 6 ⁇ m, 7 ⁇ m, 8 ⁇ m, 9 ⁇ m, 10 ⁇ m, 11 ⁇ m, 12 ⁇ m, 13 ⁇ m, 14 ⁇ m, 15 ⁇ m, 16 ⁇ m, 17 ⁇ m, 18 ⁇ m, 19 ⁇ m, 20 ⁇ m, 25 ⁇ m, 30 ⁇ m, 35 ⁇ m, 40 ⁇ m, 45 ⁇ m, 50 ⁇ m, 55 ⁇ m, 60 ⁇ m, 65 ⁇ m, 70 ⁇ m, 75 ⁇ m, 80 ⁇ m, 85 ⁇ m, 90 ⁇ m, 100 ⁇ m, 120 ⁇ m, 140 ⁇ m, 160 ⁇ m, 180 ⁇ m, 200 ⁇ m, 250 ⁇ m, 300 ⁇ m, 350 ⁇ m, 400 ⁇ m, 450 ⁇ m, 500 ⁇ m, 550 ⁇
• the bead can include a second cross-section of between about 1 ⁇ m and 500 m, such as between about 1 ⁇ m and 100 ⁇ m, between about 100 ⁇ m and 200 ⁇ m, between about 200 ⁇ m and 300 ⁇ m, between about 300 ⁇ m and 400 ⁇ m, or between about 400 ⁇ m and 500 ⁇ m.
• the bead can include a second cross-section of between about 1 ⁇ m and 100 ⁇ m.
• beads can be of a nanometer scale (e.g., beads can have a diameter or maximum cross-sectional dimension of about 100 nanometers (nm) to about 900 nanometers (nm) (e.g., 850 nm or less, 800 nm or less, 750 nm or less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less).
• nm nanometer scale
• a plurality of beads can have an average diameter or average maximum cross-sectional dimension of about 100 nanometers (nm) to about 900 nanometers (nm) (e.g., 850 nm or less, 800 nm or less, 750 nm or less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less).
• a bead has a diameter or size that is about the size of a single cell (e.g., a single cell under evaluation).
• Beads can be of uniform size or heterogeneous size. “Polydispersity” generally refers to heterogeneity of sizes of molecules or particles.
• beads can be provided as a population or plurality of beads having a relatively monodisperse size distribution. Where it can be desirable to provide relatively consistent amounts of reagents, maintaining relatively consistent bead characteristics, such as size, can contribute to the overall consistency.
• the beads provided herein can have size distributions that have a coefficient of variation in their cross-sectional dimensions of less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, or lower.
• a plurality of beads provided herein has a polydispersity index of less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or lower.
• an array includes a plurality of features.
• an array includes between 4,000 and 10,000 features, or any range within 4,000 to 6000 features.
• an array includes between 4,000 to 4,400 features, 4,000 to 4,800 features, 4,000 to 5,200 features, 4,000 to 5,600 features, 5,600 to 6,000 features, 5,200 to 6,000 features, 4,800 to 6,000 features, or 4,400 to 6,000 features.
• the array includes between 4,100 and 5,900 features, between 4,200 and 5,800 features, between 4,300 and 5,700 features, between 4,400 and 5,600 features, between 4,500 and 5,500 features, between 4,600 and 5,400 features, between 4,700 and 5,300 features, between 4,800 and 5,200 features, between 4,900 and 5,100 features, or any range within the disclosed sub-ranges.
• the array can include about 4,000 features, about 4,200 features, about 4,400 features, about 4,800 features, about 5,000 features, about 5,200 features, about 5,400 features, about 5,600 features, or about 6,000 features.
• the array comprises at least 4,000 features. In some embodiments, the array includes approximately 5,000 features.
• features within an array have an irregular arrangement or relationship to one another, such that no discernable pattern or regularity is evident in the geometrical spacing relationships among the features.
• features within an array may be positioned randomly with respect to one another.
• features within an array may be positioned irregularly, but the spacings may be selected deterministically to ensure that the resulting arrangement of features is irregular.
• features within an array are positioned regularly with respect to one another to form a pattern.
• patterns of features can be implemented in arrays. Examples of such patterns include, but are not limited to, square arrays of features, rectangular arrays of features, hexagonal arrays of features (including hexagonal close-packed arrays), radial arrays of features, spiral arrays of features, triangular arrays of features, and more generally, any array in which adjacent features in the array are reached from one another by regular increments in linear and/or angular coordinate dimensions.
• features within an array are positioned with a degree of regularity with respect to one another such that the array of features is neither perfectly regular nor perfectly irregular (i.e., the array is “partially regular”).
• adjacent features in an array can be separated by a displacement in one or more linear and/or angular coordinate dimensions that is 10% or more (e.g., 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more) of an average displacement or a nominal displacement between adjacent features in the array.
• the distribution of displacements (linear and/or angular) between adjacent features in an array has a full-width at half-maximum of between 0% and 200% (e.g., between 0% and 100%, between 0% and 75%, between 0% and 50%, between 0% and 25%, between 0% and 15%, between 0% and 10%) of an average displacement or nominal displacement between adjacent features in the array.
• arrays of features can have a variable geometry.
• a first subset of features in an array can be arranged according to a first geometrical pattern
• a second subset of features in the array can be arranged according to a second geometrical pattern that is different from the first pattern.
• Any of the patterns described above can correspond to the first and/or second geometrical patterns, for example.
• arrays of different feature densities can be prepared by adjusting the spacing between adjacent features in the array.
• the geometric center-to-center spacing between adjacent features in an array is between 100 nm and 100 ⁇ m.
• the center-to-center spacing can be between 20 ⁇ m to 40 ⁇ m, 20 ⁇ m to 60 ⁇ m, 20 ⁇ m to 80 ⁇ m, 80 ⁇ m to 100 ⁇ m, 60 ⁇ m to 100 ⁇ m, or 40 ⁇ m to 100 ⁇ m.
• the center-to-center spacing between adjacent array features is between 30 ⁇ m and 100 ⁇ m, 40 ⁇ m and 90 ⁇ m, 50 ⁇ m and 80 ⁇ m, 60 ⁇ m and 70 ⁇ m, 80 ⁇ m and 120 ⁇ m, or any range within the disclosed sub-ranges. In some embodiments, the center-to-center spacing between adjacent array features of a feature of an array is approximately 65 ⁇ m.
• an array of features can have a spatially varying resolution.
• an array with a spatially varying resolution is an array in which the center-to-center spacing (along linear, angular, or both linear and angular coordinate dimensions) between adjacent features in the array varies.
• Such arrays can be useful in a variety of applications.
• the sample can be selectively associated with the portion of the array that corresponds approximately to the desired spatial resolution of the measurement.
• Arrays of spatially varying resolution can be implemented in a variety of ways.
• the center-to-center spacing between adjacent features in the array varies continuously along one or more linear and/or angular coordinate directions.
• the spacing between successive rows of features, between successive columns of features, or between both successive rows and successive columns of features can vary continuously.
• arrays of spatially varying resolution can include discrete domains with populations of features. Within each domain, adjacent features can have regular center-to-center spacings.
• an array can include a first domain within which adjacent features are spaced from one another along linear and/or angular coordinate dimensions by a first set of uniform coordinate displacements, and a second domain within which adjacent features are spaced from one another along linear and/or angular coordinate dimensions by a second set of uniform coordinate displacements.
• the first and second sets of displacements differ in at least one coordinate displacement, such that adjacent features in the two domains are spaced differently, and the resolution of the array in the first domain is therefore different from the resolution of the array in the second domain.
• the center-to-center spacing of array features can be sufficiently small such that array features are effectively positioned continuously or nearly continuously along one or more array dimensions, with little or no displacement between array features along those dimensions.
• the displacement between adjacent oligonucleotides can be very small—effectively, the molecular width of a single oligonucleotide.
• each oligonucleotide can include a distinct spatial barcode such that the spatial location of each oligonucleotide in the array can be determined during sample analysis. Arrays of this type can have very high spatial resolution, but may only include a single oligonucleotide corresponding to each distinct spatial location in a sample.
• the size of the array (which corresponds to the maximum dimension of the smallest boundary that encloses all features in the array along one coordinate direction) can be selected as desired, based on criteria such as the size of the sample, the feature sizes, and the density of capture probes within each feature.
• the array can be a rectangular or square array for which the maximum array dimension along each coordinate direction is 10 mm or less (e.g., 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less).
• a square array of features can have dimensions of 8 mm by 8 mm, 7 mm by 7 mm, 5 mm by 5 mm, or be smaller than 5 mm by 5 mm.
• analytes can be captured when contacting a biological sample with, e.g., a substrate comprising capture probes (e.g., substrate with capture probes embedded, spotted, printed on the substrate or a substrate with features (e.g., beads, wells) comprising capture probes).
• a substrate comprising capture probes (e.g., substrate with capture probes embedded, spotted, printed on the substrate or a substrate with features (e.g., beads, wells) comprising capture probes).
• a biological sample with a substrate comprising features refers to any contact (e.g., direct or indirect) such that capture probes can interact (e.g., capture) with analytes from the biological sample.
• the substrate may be near or adjacent to the biological sample without direct physical contact, yet capable of capturing analytes from the biological sample.
• the biological sample is in direct physical contact with the substrate.
• the biological sample is in indirect physical contact with the substrate.
• a liquid layer may be between the biological sample and the substrate.
• the analytes diffuse through the liquid layer.
• the capture probes diffuse through the liquid layer.
• reagents may be delivered via the liquid layer between the biological sample and the substrate.
• indirect physical contact may be the presence of a second substrate (e.g., a hydrogel, a film, a porous membrane) between the biological sample and the first substrate comprising features with capture probes.
• reagents may be delivered by the second substrate to the biological sample.
• a diffusion-resistant medium can be used.
• molecular diffusion of biological analytes occurs in all directions, including toward the capture probes (i.e. toward the spatially-barcoded array), and away from the capture probes (i.e. into the bulk solution).
• Increasing diffusion toward the spatially-barcoded array reduces analyte diffusion away from the spatially-barcoded array and increases the capturing efficiency of the capture probes.
• a biological sample is placed on the top of a spatially-barcoded substrate and a diffusion-resistant medium is placed on top of the biological sample.
• the diffusion-resistant medium can be placed onto an array that has been placed in contact with a biological sample.
• the diffusion-resistant medium and spatially-labelled array are the same component.
• the diffusion-resistant medium can contain spatially-labelled capture probes within or on the diffusion-resistant medium (e.g., coverslip, slide, hydrogel, or membrane).
• a sample is placed on a substrate and a diffusion-resistant medium is placed on top of the biological sample.
• a spatially-barcoded capture probe array can be placed in close proximity over the diffusion-resistant medium.
• a diffusion-resistant medium may be sandwiched between a spatially-labelled array and a sample on a substrate.
• the diffusion-resistant medium is disposed or spotted onto the sample.
• the diffusion-resistant medium is placed in close proximity to the sample.
• the diffusion-resistant medium can be any material known to limit diffusivity of biological analytes.
• the diffusion-resistant medium can be a solid lid (e.g., coverslip or glass slide).
• the diffusion-resistant medium may be made of glass, silicon, paper, hydrogel polymer monoliths, or other material.
• the glass side can be an acrylated glass slide.
• the diffusion-resistant medium is a porous membrane.
• the material may be naturally porous.
• the material may have pores or wells etched into solid material.
• the pore size can be manipulated to minimize loss of target analytes.
• the membrane chemistry can be manipulated to minimize loss of target analytes.
• the diffusion-resistant medium i.e. hydrogel
• the diffusion-resistant medium can be any material known to limit diffusivity of poly(A) transcripts.
• the diffusion-resistant medium can be any material known to limit the diffusivity of proteins.
• the diffusion-resistant medium can be any material know to limit the diffusivity of macromolecular constituents.
• a diffusion-resistant medium includes one or more diffusion-resistant media.
• one or more diffusion-resistant media can be combined in a variety of ways prior to placing the media in contact with a biological sample including, without limitation, coating, layering, or spotting.
• a hydrogel can be placed onto a biological sample followed by placement of a lid (e.g., glass slide) on top of the hydrogel.
• a force e.g., hydrodynamic pressure, ultrasonic vibration, solute contrasts, microwave radiation, vascular circulation, or other electrical, mechanical, magnetic, centrifugal, and/or thermal forces
• a force e.g., hydrodynamic pressure, ultrasonic vibration, solute contrasts, microwave radiation, vascular circulation, or other electrical, mechanical, magnetic, centrifugal, and/or thermal forces
• one or more forces and one or more diffusion-resistant media are used to control diffusion and enhance capture.
• a centrifugal force and a glass slide can used contemporaneously. Any of a variety of combinations of a force and a diffusion-resistant medium can be used to control or mitigate diffusion and enhance analyte capture.
• the diffusion-resistant medium along with the spatially-barcoded array and sample, is submerged in a bulk solution.
• the bulk solution includes permeabilization reagents.
• the diffusion-resistant medium includes at least one permeabilization reagent.
• the diffusion-resistant medium i.e. hydrogel
• the diffusion-resistant medium is soaked in permeabilization reagents before contacting the diffusion-resistant medium to the sample.
• the diffusion-resistant medium can include wells (e.g., micro-, nano-, or picowells) containing a permeabilization buffer or reagents.
• the diffusion-resistant medium can include permeabilization reagents.
• the diffusion-resistant medium can contain dried reagents or monomers to deliver permeabilization reagents when the diffusion-resistant medium is applied to a biological sample.
• the diffusion-resistant medium is added to the spatially-barcoded array and sample assembly before the assembly is submerged in a bulk solution.
• the diffusion-resistant medium is added to the spatially-barcoded array and sample assembly after the sample has been exposed to permeabilization reagents.
• the permeabilization reagents are flowed through a microfluidic chamber or channel over the diffusion-resistant medium. In some embodiments, the flow controls the sample's access to the permeabilization reagents.
• the target analytes diffuse out of the sample and toward a bulk solution and get embedded in a spatially-labelled capture probe-embedded diffusion-resistant medium.
• a free solution is sandwiched between the biological sample and a diffusion-resistant medium.
• FIG. 13 is an illustration of an exemplary use of a diffusion-resistant medium.
• a diffusion-resistant medium 1302 can be contacted with a sample 1303 .
• a glass slide 1304 is populated with spatially-barcoded capture probes 1306 , and the sample 1303 , 1305 is contacted with the array 1304 , 1306 .
• a diffusion-resistant medium 1302 can be applied to the sample 1303 , wherein the sample 1303 is sandwiched between a diffusion-resistant medium 1302 and a capture probe coated slide 1304 .
• the diffusion-resistant medium/lid 1302 directs migration of the analytes 1305 toward the capture probes 1306 by reducing diffusion of the analytes out into the medium.
• the lid may contain permeabilization reagents.
• Capture probes on the substrate interact with released analytes through a capture domain, described elsewhere, to capture analytes.
• certain steps are performed to enhance the transfer or capture of analytes by the capture probes of the array.
• modifications include, but are not limited to, adjusting conditions for contacting the substrate with a biological sample (e.g., time, temperature, orientation, pH levels, pre-treating of biological samples, etc.), using force to transport analytes (e.g., electrophoretic, centrifugal, mechanical, etc.), performing amplification reactions to increase the amount of biological analytes (e.g., PCR amplification, in situ amplification, clonal amplification), and/or using labeled probes for detecting of amplicons and barcodes.
• a biological sample e.g., time, temperature, orientation, pH levels, pre-treating of biological samples, etc.
• force to transport analytes e.g., electrophoretic, centrifugal, mechanical, etc.
• performing amplification reactions to increase the amount of biological analytes (e.g., PCR amplification, in situ amplification, clonal amplification), and/or using labeled probes for detecting of
• capture of analytes is facilitated by treating the biological sample with permeabilization reagents. If a biological sample is not permeabilized sufficiently, the amount of analyte captured on the substrate can be too low to enable adequate analysis. Conversely, if the biological sample is too permeable, the analyte can diffuse away from its origin in the biological sample, such that the relative spatial relationship of the analytes within the biological sample is lost. Hence, a balance between permeabilizing the biological sample enough to obtain good signal intensity while still maintaining the spatial resolution of the analyte distribution in the biological sample is desired.
• Methods of preparing biological samples to facilitation are known in the art and can be modified depending on the biological sample and how the biological sample is prepared (e.g., fresh frozen, FFPE, etc.).
• analytes can be migrated from a sample to a substrate.
• Methods for facilitating migration can be passive (e.g., diffusion) and/or active (e.g., electrophoretic migration of nucleic acids).
• passive migration can include simple diffusion and osmotic pressure created by the rehydration of dehydrated objects.
• Diffusion is movement of untethered objects toward equilibrium. Therefore, when there is a region of high object concentration and a region of low object concentration, the object (capture probe, the analyte, etc.) moves to an area of lower concentration. In some embodiments, untethered analytes move down a concentration gradient.
• different reagents may be added to the biological sample, such that the biological sample is rehydrated while improving capture of analytes.
• the biological sample can be rehydrated with permeabilization reagents.
• the biological sample can be rehydrated with a staining solution (e.g., hematoxylin and eosin stain).
• an analyte in a cell or a biological sample can be transported (e.g., passively or actively) to a capture probe (e.g., a capture probe affixed to a solid surface).
• a capture probe e.g., a capture probe affixed to a solid surface
• analytes in a cell or a biological sample can be transported to a capture probe (e.g., an immobilized capture probe) using an electric field (e.g., using electrophoresis), a pressure gradient, fluid flow, a chemical concentration gradient, a temperature gradient, and/or a magnetic field.
• analytes can be transported through, e.g., a gel (e.g., hydrogel matrix), a fluid, or a permeabilized cell, to a capture probe (e.g., an immobilized capture probe).
• an electrophoretic field can be applied to analytes to facilitate migration of the analytes towards a capture probe.
• a sample contacts a substrate and capture probes fixed on a substrate (e.g., a slide, cover slip, or bead), and an electric current is applied to promote the directional migration of charged analytes towards the capture probes fixed on the substrate.
• An electrophoresis assembly where a cell or a biological sample is in contact with a cathode and capture probes (e.g., capture probes fixed on a substrate), and where the capture probes (e.g., capture probes fixed on a substrate) is in contact with the cell or biological sample and an anode, can be used to apply the current.
• Electrophoretic transfer of analytes can be performed while retaining the relative spatial alignment of the analytes in the sample.
• an analyte captured by the capture probes e.g., capture probes fixed on a substrate
• Applying an electrophoretic field to analytes can also result in an increase in temperature (e.g., heat).
• the increased temperature e.g., heat
• a spatially-addressable microelectrode array is used for spatially-constrained capture of at least one charged analyte of interest by a capture probe.
• the microelectrode array can be configured to include a high density of discrete sites having a small area for applying an electric field to promote the migration of charged analyte(s) of interest.
• electrophoretic capture can be performed on a region of interest using a spatially-addressable microelectrode array.
• a high density of discrete sites on a microelectrode array can be used for small device.
• the surface can include any suitable density of discrete sites (e.g., a density suitable for processing the sample on the conductive substrate in a given amount of time). In an embodiment, the surface has a density of discrete sites greater than or equal to about 500 sites per 1 mm 2 .
• the surface has a density of discrete sites of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 20,000, about 40,000, about 60,000, about 80,000, about 100,000, or about 500,000 sites per 1 mm 2 .
• the surface has a density of discrete sites of at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1,000, at least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, at least about 6,000, at least about 7,000, at least about 8,000, at least about 9,000, at least about 10,000, at least about 20,000, at least about 40,000, at least about 60,000, at least about 80,000, at least about 100,000, or at least about 500,000 sites per 1 mm 2 .
• FIG. 14 A and FIG. 14 B Schematics illustrating an electrophoretic transfer system configured to direct transcript analytes toward a spatially-barcoded capture probe array are shown in FIG. 14 A and FIG. 14 B .
• a sample 1402 is sandwiched between the cathode 1401 and the spatially-barcoded capture probe array 1404 , 1405 , and the spatially-barcoded capture probe array 1404 , 1405 is sandwiched between the sample 1402 and the anode 1403 , such that the sample 1402 , 1406 is in contact with the spatially-barcoded capture probes 1407 .
• an electric field is applied to the electrophoretic transfer system, negatively charged mRNA analytes 1406 will be pulled toward the positively charged anode 1403 and into the spatially-barcoded array 1404 , 1405 containing the spatially-barcoded capture probes 1407 .
• the spatially-barcoded capture probes 1407 then interact with/hybridize with/immobilize the mRNA target analytes 1406 , making the analyte capture more efficient.
• the electrophoretic system set-up may change depending on the target analyte.
• proteins may be positive, negative, neutral, or polar depending on the protein as well as other factors (e.g. isoelectric point, solubility, etc.).
• the skilled practitioner has the knowledge and experience to arrange the electrophoretic transfer system to facilitate capture of a particular target analyte.
• FIG. 15 is an illustration showing an exemplary workflow protocol utilizing an electrophoretic transfer system.
• Panel A depicts a flexible spatially-barcoded feature array being contacted with a sample.
• the sample can be a flexible array, wherein the array is immobilized on a hydrogel, membrane, or other flexible substrate.
• Panel B depicts contact of the array with the sample and imaging of the array-sample assembly.
• the image of the sample/array assembly can be used to verify sample placement, choose a region of interest, or any other reason for imaging a sample on an array as described herein.
• Panel C depicts application of an electric field using an electrophoretic transfer system to aid in efficient capture of a target analyte.
• Panel D depicts application of reverse transcription reagents and first strand cDNA synthesis of the captured target analytes.
• Panel E depicts array removal and preparation for library construction (Panel F) and next-generation sequencing (Panel G).
• a biological sample can have regions that show morphological feature(s) that may indicate the presence of disease or the development of a disease phenotype.
• morphological features at a specific site within a tumor biopsy sample can indicate the aggressiveness, therapeutic resistance, metastatic potential, migration, stage, diagnosis, and/or prognosis of cancer in a subject.
• a change in the morphological features at a specific site within a tumor biopsy sample often correlate with a change in the level or expression of an analyte in a cell within the specific site, which can, in turn, be used to provide information regarding the aggressiveness, therapeutic resistance, metastatic potential, migration, stage, diagnosis, and/or prognosis of cancer in a subject.
• a region or area within a biological sample that is selected for specific analysis is often described as “a region of interest.”
• a region of interest in a biological sample can be used to analyze a specific area of interest within a biological sample, and thereby, focus experimentation and data gathering to a specific region of a biological sample (rather than an entire biological sample). This results in increased time efficiency of the analysis of a biological sample.
• a region of interest can be identified in a biological sample using a variety of different techniques, e.g., expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy, confocal microscopy, and visual identification (e.g., by eye), and combinations thereof.
• the staining and imaging of a biological sample can be performed to identify a region of interest.
• the region of interest can correspond to a specific structure of cytoarchitecture.
• a biological sample can be stained prior to visualization to provide contrast between the different regions of the biological sample.
• the type of stain can be chosen depending on the type of biological sample and the region of the cells to be stained.
• more than one stain can be used to visualize different aspects of the biological sample, e.g., different regions of the sample, specific cell structures (e.g. organelles), or different cell types.
• the biological sample can be visualized or imaged without staining the biological sample.
• imaging can be performed using one or more fiducial markers, i.e., objects placed in the field of view of an imaging system which appear in the image produced.
• Fiducial markers are typically used as a point of reference or measurement scale.
• Fiducial markers can include, but are not limited to, detectable labels such as fluorescent, radioactive, chemiluminescent, and colorimetric labels. The use of fiducial markers to stabilize and orient biological samples is described, for example, in Carter et al., Applied Optics 46:421-427, 2007), the entire contents of which are incorporated herein by reference.
• a fiducial marker can be a physical particle (e.g., a nanoparticle, a microsphere, a nanosphere, a bead, or any of the other exemplary physical particles described herein or known in the art).
• a fiducial marker can be present on a substrate to provide orientation of the biological sample.
• a microsphere can be coupled to a substrate to aid in orientation of the biological sample.
• a microsphere coupled to a substrate can produce an optical signal (e.g., fluorescence).
• a microsphere can be attached to a portion (e.g., corner) of an array in a specific pattern or design (e.g., hexagonal design) to aid in orientation of a biological sample on an array of features on the substrate.
• a quantum dot can be coupled to the substrate to aid in the orientation of the biological sample.
• a quantum dot coupled to a substrate can produce an optical signal.
• a fiducial marker can be an immobilized molecule with which a detectable signal molecule can interact to generate a signal.
• a marker nucleic acid can be linked or coupled to a chemical moiety capable of fluorescing when subjected to light of a specific wavelength (or range of wavelengths).
• Such a marker nucleic acid molecule can be contacted with an array before, contemporaneously with, or after the tissue sample is stained to visualize or image the tissue section.
• fiducial markers are included to facilitate the orientation of a tissue sample or an image thereof in relation to an immobilized capture probes on a substrate. Any number of methods for marking an array can be used such that a marker is detectable only when a tissue section is imaged.
• a molecule e.g. a fluorescent molecule that generates a signal
• Markers can be provided on a substrate in a pattern (e.g., an edge, one or more rows, one or more lines, etc.).
• a fiducial marker can be randomly placed in the field of view.
• an oligonucleotide containing a fluorophore can be randomly printed, stamped, synthesized, or attached to a substrate (e.g., a glass slide) at a random position on the substrate.
• a tissue section can be contacted with the substrate such that the oligonucleotide containing the fluorophore contacts, or is in proximity to, a cell from the tissue section or a component of the cell (e.g., an mRNA or DNA molecule).
• fiducial markers can be precisely placed in the field of view (e.g., at known locations on a substrate).
• a fiducial marker can be stamped, attached, or synthesized on the substrate and contacted with a biological sample.
• an image of the sample and the fiducial marker is taken, and the position of the fiducial marker on the substrate can be confirmed by viewing the image.
• a fiducial marker can be an immobilized molecule (e.g., a physical particle) attached to the substrate.
• a fiducial marker can be a nanoparticle, e.g., a nanorod, a nanowire, a nanocube, a nanopyramid, or a spherical nanoparticle.
• the nanoparticle can be made of a heavy metal (e.g., gold).
• the nanoparticle can be made from diamond.
• the fiducial marker can be visible by eye.
• any of the fiducial markers described herein can be located at a portion (e.g., corner) of an array in a specific pattern or design (e.g., hexagonal design) to aid in orientation of a biological sample on an array of features on the substrate.
• the fiducial markers located at a portion (e.g., corner) of an array e.g., an array on a substrate
• the fiducial markers located at the corners of the array e.g., an array on a substrate
• fiducial markers can surround the array. In some embodiments the fiducial markers allow for detection of, e.g., mirroring. In some embodiments, the fiducial markers may completely surround the array. In some embodiments, the fiducial markers may not completely surround the array. In some embodiments, the fiducial markers identify the corners of the array. In some embodiments, one or more fiducial markers identify the center of the array. In some embodiments, the fiducial markers comprise patterned spots, wherein the diameter of one or more patterned spot fiducial markers is approximately 100 micrometers. The diameter of the fiducial markers can be any useful diameter including, but not limited to, 50 micrometers to 500 micrometers in diameter.
• the fiducial markers may be arranged in such a way that the center of one fiducial marker is between 100 micrometers and 200 micrometers from the center of one or more other fiducial markers surrounding the array.
• the array with the surrounding fiducial markers is approximately 8 mm by 8 mm. In some embodiments, the array without the surrounding fiducial markers is smaller than 8 mm by 50 mm.
• an array can be enclosed within a frame.
• the perimeter of an array can have fiducial markers such that the array is enclosed, or substantially enclosed.
• the perimeter of an array can be fiducial markers (e.g., any fiducial marker described herein).
• the perimeter of an array can be uniform.
• the fiducial markings can connect, or substantially connect, consecutive corners of an array in such a fashion that the non-corner portion of the array perimeter is the same on all sides (e.g., four sides) of the array.
• the fiducial markers attached to the non-corner portions of the perimeter can be pattered or designed to aid in the orientation of the biological sample on the array.
• the particles attached to the non-corner portions of the perimeter can be patterned or designed in at least 1, at least 2, at least 3, or at least 4 patterns.
• the patterns can have at least 2, at least 3, or at least 4 unique patterns of fiducial markings on the non-corner portion of the array perimeter.
• an array can include at least two fiducial markers (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 fiducial markers or more (e.g., several hundred, several thousand, or tens of thousands of fiducial markers)) in distinct positions on the surface of a substrate.
• Fiducial markers can be provided on a substrate in a pattern (e.g., an edge, one or more rows, one or more lines, etc.).
• staining and imaging a biological sample prior to contacting the biological sample with a spatial array is performed to select samples for spatial analysis.
• the staining includes applying a fiducial marker as described above, including fluorescent, radioactive, chemiluminescent, or colorimetric detectable markers.
• the staining and imaging of biological samples allows the user to identify the specific sample (or region of interest) the user wishes to assess.
• a lookup table can be used to associate one property with another property of a feature.
• properties include, e.g., locations, barcodes (e.g., nucleic acid barcode molecules), spatial barcodes, optical labels, molecular tags, and other properties.
• a lookup table can associate a nucleic acid barcode molecule with a feature.
• an optical label of a feature can permit associating the feature with a biological particle (e.g., cell or nuclei).
• the association of a feature with a biological particle can further permit associating a nucleic acid sequence of a nucleic acid molecule of the biological particle to one or more physical properties of the biological particle (e.g., a type of a cell or a location of the cell).
• the optical label can be used to determine the location of a feature, thus associating the location of the feature with the barcode sequence of the feature.
• Subsequent analysis can associate the barcode sequence and the analyte from the sample. Accordingly, based on the relationship between the location and the barcode sequence, the location of the biological analyte can be determined (e.g., in a specific type of cell or in a cell at a specific location of the biological sample).
• a feature can have a plurality of nucleic acid barcode molecules attached thereto.
• the plurality of nucleic acid barcode molecules can include barcode sequences.
• the plurality of nucleic acid molecules attached to a given feature can have the same barcode sequences, or two or more different barcode sequences. Different barcode sequences can be used to provide improved spatial location accuracy.
• a substrate is treated in order to minimize or reduce non-specific analyte hybridization within or between features.
• treatment can include coating the substrate with a hydrogel, film, and/or membrane that creates a physical barrier to non-specific hybridization.
• Any suitable hydrogel can be used.
• hydrogel matrices prepared according to the methods set forth in U.S. Pat. Nos. 6,391,937, 9,512,422, and 9,889,422, and U.S. Patent Application Publication Nos. U.S. 2017/0253918 and U.S. 2018/0052081 can be used. The entire contents of each of the foregoing documents are incorporated herein by reference.
• Treatment can include adding a functional group that is reactive or capable of being activated such that it becomes reactive after receiving a stimulus (e.g., photoreactive).
• Treatment can include treating with polymers having one or more physical properties (e.g., mechanical, electrical, magnetic, and/or thermal) that minimize non-specific binding (e.g., that activate a substrate at certain locations to allow analyte hybridization at those locations).
• an array (e.g., any of the exemplary arrays described herein) can be contacted with only a portion of a biological sample (e.g., a cell, a feature, or a region of interest).
• a biological sample is contacted with only a portion of an array (e.g., any of the exemplary arrays described herein).
• a portion of the array can be deactivated such that it does not interact with the analytes in the biological sample (e.g., optical deactivation, chemical deactivation, heat deactivation, or blocking of the capture probes in the array (e.g., using blocking probes)).
• a region of interest can be removed from a biological sample and then the region of interest can be contacted to the array (e.g., any of the arrays described herein).
• a region of interest can be removed from a biological sample using microsurgery, laser capture microdissection, chunking, a microtome, dicing, trypsinization, labelling, and/or fluorescence-assisted cell sorting.
• the sample can optionally be separated into single cells, cell groups, or other fragments/pieces that are smaller than the original, unfragmented sample. Each of these smaller portions of the sample can be analyzed to obtain spatially-resolved analyte information for the sample.
• one method for analyzing the fragments involves partitioning the fragments into individual partitions (e.g., fluid droplets), and then analyzing the contents of the partitions.
• each partition maintains separation of its own contents from the contents of other partitions.
• the partition can be a droplet in an emulsion, for example.
• a partition can include additional components, and in particular, one or more beads.
• a partition can include a single gel bead, a single cell bead, or both a single cell bead and single gel bead.
• a partition can also include one or more reagents.
• Unique identifiers such as barcodes, can be injected into the droplets previous to, subsequent to, or concurrently with droplet generation, such as via a microcapsule (e.g., bead).
• Microfluidic channel networks e.g., on a chip
• Alternative mechanisms can also be employed in the partitioning of individual biological particles, including porous membranes through which aqueous mixtures of cells are extruded into non-aqueous fluids.
• the partitions can be flowable within fluid streams.
• the partitions can include, for example, micro-vesicles that have an outer barrier surrounding an inner fluid center or core.
• the partitions can include a porous matrix that is capable of entraining and/or retaining materials within its matrix.
• the partitions can be droplets of a first phase within a second phase, wherein the first and second phases are immiscible.
• the partitions can be droplets of aqueous fluid within a non-aqueous continuous phase (e.g., oil phase).
• the partitions can be droplets of a non-aqueous fluid within an aqueous phase.
• the partitions can be provided in a water-in-oil emulsion or oil-in-water emulsion.
• a variety of different vessels are described in, for example, U.S. Patent Application Publication No. 2014/0155295, the entire contents of which are incorporated herein by reference.
• Emulsion systems for creating stable droplets in non-aqueous or oil continuous phases are described, for example, in U.S. Patent Application Publication No. 2010/0105112, the entire contents of which are incorporated herein by reference.
• allocating individual particles to discrete partitions can be accomplished, for example, by introducing a flowing stream of particles in an aqueous fluid into a flowing stream of a non-aqueous fluid, such that droplets are generated at the junction of the two streams.
• Fluid properties e.g., fluid flow rates, fluid viscosities, etc.
• particle properties e.g., volume fraction, particle size, particle concentration, etc.
• microfluidic architectures e.g., channel geometry, etc.
• partition occupancy can be controlled by providing the aqueous stream at a certain concentration and/or flow rate of analytes.
• the relative flow rates of the immiscible fluids can be selected such that, on average, the partitions can contain less than one analyte per partition to ensure that those partitions that are occupied are primarily singly occupied.
• partitions among a plurality of partitions can contain at most one analyte.
• the various parameters e.g., fluid properties, particle properties, microfluidic architectures, etc.
• the flows and channel architectures can be controlled as to ensure a given number of singly occupied partitions, less than a certain level of unoccupied partitions and/or less than a certain level of multiply occupied partitions.
• the channel segments described herein can be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, tubing, manifolds, or fluidic components of other systems.
• the microfluidic channel structure can have a variety of geometries.
• a microfluidic channel structure can have one or more than one channel junction.
• a microfluidic channel structure can have 2, 3, 4, or 5 channel segments each carrying particles that meet at a channel junction.
• Fluid can be directed to flow along one or more channels or reservoirs via one or more fluid flow units.
• a fluid flow unit can include compressors (e.g., providing positive pressure), pumps (e.g., providing negative pressure), actuators, and the like to control flow of the fluid. Fluid can also or otherwise be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary, and/or gravity flow.
• a partition can include one or more unique identifiers, such as barcodes.
• Barcodes can be previously, subsequently, or concurrently delivered to the partitions that hold the compartmentalized or partitioned biological particle.
• barcodes can be injected into droplets previous to, subsequent to, or concurrently with droplet generation.
• the delivery of the barcodes to a particular partition allows for the later attribution of the characteristics of the individual biological particle to the particular partition.
• Barcodes can be delivered, for example on a nucleic acid molecule (e.g., an oligonucleotide), to a partition via any suitable mechanism.
• Barcoded nucleic acid molecules can be delivered to a partition via a microcapsule.
• a microcapsule in some instances, can include a bead.
• barcoded nucleic acid molecules can be initially associated with the microcapsule and then released from the microcapsule. Release of the barcoded nucleic acid molecules can be passive (e.g., by diffusion out of the microcapsule). In addition or alternatively, release from the microcapsule can be upon application of a stimulus which allows the barcoded nucleic acid nucleic acid molecules to dissociate or to be released from the microcapsule. Such stimulus can disrupt the microcapsule, an interaction that couples the barcoded nucleic acid molecules to or within the microcapsule, or both.
• Such stimulus can include, for example, a thermal stimulus, photo-stimulus, chemical stimulus (e.g., change in pH or use of a reducing agent(s)), a mechanical stimulus, a radiation stimulus; a biological stimulus (e.g., enzyme), or any combination thereof.
• chemical stimulus e.g., change in pH or use of a reducing agent(s)
• mechanical stimulus e.g., change in pH or use of a reducing agent(s)
• a radiation stimulus e.g., a radiation stimulus
• a biological stimulus e.g., enzyme
• one more barcodes can be introduced into a partition as part of the analyte.
• barcodes can be bound to the analyte directly, or can form part of a capture probe or analyte capture agent that is hybridized to, conjugated to, or otherwise associated with an analyte, such that when the analyte is introduced into the partition, the barcode(s) are introduced as well.
• FIG. 16 shows an example of a microfluidical channel structure for partitioning individual analytes (e.g., cells) into discrete partitions.
• FIG. 16 shows an example of a microfluidic channel structure for partitioning individual analytes (e.g., cells) into discrete partitions.
• the channel structure can include channel segments 1601 , 1602 , 1603 , and 1604 communicating at a channel junction 1605 .
• a first aqueous fluid 1606 that includes suspended biological particles (or cells) 1607 may be transported along channel segment 1601 into junction 1605
• a second fluid 1608 that is immiscible with the aqueous fluid 1606 is delivered to the junction 1605 from each of channel segments 1602 and 1603 to create discrete droplets 1609 , 1610 of the first aqueous fluid 1606 flowing into channel segment 1604 , and flowing away from junction 1605 .
• the channel segment 1604 may be fluidically coupled to an outlet reservoir where the discrete droplets can be stored and/or harvested.
• a discrete droplet generated may include an individual biological particle 1607 (such as droplets 1609 ).
• a discrete droplet generated may include more than one individual biological particle 1607 .
• a discrete droplet may contain no biological particle 1607 (such as droplet 1610 ).
• Each discrete partition may maintain separation of its own contents (e.g., individual biological particle 1607 ) from the contents of other partitions.
• FIG. 17 A shows another example of a microfluidic channel structure 1700 for delivering beads to droplets.
• the channel structure includes channel segments 1701 , 1702 , 1703 , 1704 and 1705 communicating at a channel junction 1706 .
• the channel segment 1701 can transport an aqueous fluid 1707 that includes a plurality of beads 1708 along the channel segment 1701 into junction 1706 .
• the plurality of beads 1708 can be sourced from a suspension of beads.
• the channel segment 1701 can be connected to a reservoir that includes an aqueous suspension of beads 1708 .
• the channel segment 1702 can transport the aqueous fluid 1707 that includes a plurality of particles 1709 (e.g., cells) along the channel segment 1702 into junction 1706 .
• the aqueous fluid 1707 in either the first channel segment 1701 or the second channel segment 1702 , or in both segments can include one or more reagents, as further described below.
• a second fluid 1710 that is immiscible with the aqueous fluid 1707 can be delivered to the junction 1706 from each of channel segments 1703 and 1704 .
• the aqueous fluid 1707 can be partitioned as discrete droplets 1711 in the second fluid 1710 and flow away from the junction 1706 along channel segment 1705 .
• the channel segment 1705 can deliver the discrete droplets to an outlet reservoir fluidly coupled to the channel segment 1705 , where they can be harvested.
• the channel segments 1701 and 1702 can meet at another junction upstream of the junction 1706 .
• beads and biological particles can form a mixture that is directed along another channel to the junction 1706 to yield droplets 1711 .
• the mixture can provide the beads and biological particles in an alternating fashion, such that, for example, a droplet includes a single bead and a single biological particle.
• the second fluid 1710 can include an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets 1711 .
• an oil such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets 1711 .
• the partitions described herein can include small volumes, for example, less than about 10 microliters ( ⁇ L), 5 ⁇ L, 1 ⁇ L, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.
• droplets with beads were formed at the junction of different fluid streams.
• droplets can be formed by gravity-based partitioning methods.
• FIG. 17 B shows a cross-section view of another example of a microfluidic channel structure 1750 with a geometric feature for controlled partitioning.
• a channel structure 1750 can include a channel segment 1752 communicating at a channel junction 1758 (or intersection) with a reservoir 1754 .
• the channel structure 1750 and one or more of its components can correspond to the channel structure 1700 and one or more of its components.
• An aqueous fluid 1760 comprising a plurality of particles 1756 may be transported along the channel segment 1752 into the junction 1758 to meet a second fluid 1762 (e.g., oil, etc.) that is immiscible with the aqueous fluid 1760 in the reservoir 1754 to create droplets 1764 of the aqueous fluid 1760 flowing into the reservoir 1754 .
• a second fluid 1762 e.g., oil, etc.
• droplets can form based on factors such as the hydrodynamic forces at the junction 1758 , relative flow rates of the two fluids 1760 , 1762 , fluid properties, and certain geometric parameters (e.g., ⁇ h, etc.) of the channel structure 1750 .
• a plurality of droplets can be collected in the reservoir 1754 by continuously injecting the aqueous fluid 1760 from the channel segment 1752 at the junction 1758 .
• a discrete droplet generated may comprise one or more particles of the plurality of particles 1756 .
• a particle may be any particle, such as a bead, cell bead, gel bead, biological particle, macromolecular constituents of biological particle, or other particles.
• a discrete droplet generated may not include any particles.
• the aqueous fluid 1760 can have a substantially uniform concentration or frequency of particles 1756 .
• the particles 1756 e.g., beads
• the frequency of particles 1756 in the channel segment 1752 may be controlled by controlling the frequency in which the particles 1756 are introduced into the channel segment 1752 and/or the relative flow rates of the fluids in the channel segment 1752 and the separate channel.
• the particles 1756 can be introduced into the channel segment 1752 from a plurality of different channels, and the frequency controlled accordingly.
• different particles may be introduced via separate channels.
• a first separate channel can introduce beads and a second separate channel can introduce biological particles into the channel segment 1752 .
• the first separate channel introducing the beads may be upstream or downstream of the second separate channel introducing the biological particles.
• the second fluid 1762 may not be subjected to and/or directed to any flow in or out of the reservoir 1754 .
• the second fluid 1762 may be substantially stationary in the reservoir 1754 .
• the second fluid 1762 may be subjected to flow within the reservoir 1754 , but not in or out of the reservoir 1754 , such as via application of pressure to the reservoir 1754 and/or as affected by the incoming flow of the aqueous fluid 1760 at the junction 1758 .
• the second fluid 1762 may be subjected and/or directed to flow in or out of the reservoir 1754 .
• the reservoir 1754 can be a channel directing the second fluid 1762 from upstream to downstream, transporting the generated droplets.
• the channel structure 1750 at or near the junction 1758 may have certain geometric features that at least partly determine the sizes and/or shapes of the droplets formed by the channel structure 1750 .
• the channel segment 1752 can have a first cross-section height, h1, and the reservoir 1754 can have a second cross-section height, h2.
• the first cross-section height, h1, and the second cross-section height, h2, may be different, such that at the junction 1758 , there is a height difference of ⁇ h.
• the second cross-section height, h2 may be greater than the first cross-section height, h1.
• the reservoir may thereafter gradually increase in cross-section height, for example, the more distant it is from the junction 1758 .
• the cross-section height of the reservoir may increase in accordance with expansion angle, ⁇ , at or near the junction 1758 .
• the height difference, ⁇ h, and/or expansion angle, p can allow the tongue (portion of the aqueous fluid 1760 leaving channel segment 1752 at junction 1758 and entering the reservoir 1754 before droplet formation) to increase in depth and facilitate decrease in curvature of the intermediately formed droplet.
• droplet size may decrease with increasing height difference and/or increasing expansion angle.
• the height difference, ⁇ h can be at least about 1 ⁇ m.
• the height difference can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 ⁇ m or more.
• the height difference can be at most about 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 ⁇ m or less.
• the expansion angle, ⁇ may be between a range of from about 0.5° to about 4°, from about 0.1° to about 10°, or from about 0° to about 90°.
• the expansion angle can be at least about 0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3° 4° 5° 6°, 7° 8°, 9° 10°, 15°, 20°, 25°, 30°, 35° 40°, 45° 50°, 55° 60°, 65°, 70°, 75° 80°, 85°, or higher.
• the expansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75° 70°, 65°, 60°, 55° 50°, 45° 40°, 35° 30°, 25°, 20°, 15°, 10°, 9°, 8°, 7° 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.
• the flow rate of the aqueous fluid 1760 entering the junction 1758 can be between about 0.04 microliters ( ⁇ L)/minute (min) and about 40 ⁇ L/min. In some instances, the flow rate of the aqueous fluid 1760 entering the junction 1758 can be between about 0.01 microliters ( ⁇ L)/minute (min) and about 100 ⁇ L/min. Alternatively, the flow rate of the aqueous fluid 1760 entering the junction 1758 can be less than about 0.01 ⁇ L/min.
• the flow rate of the aqueous fluid 1760 entering the junction 1758 can be greater than about 40 ⁇ L/min, such as 45 ⁇ L/min, 50 ⁇ L/min, 55 ⁇ L/min, 60 ⁇ L/min, 65 ⁇ L/min, 70 ⁇ L/min, 75 ⁇ L/min, 80 ⁇ L/min, 85 ⁇ L/min, 90 ⁇ L/min, 95 ⁇ L/min, 100 ⁇ L/min, 110 ⁇ L/min, 120 ⁇ L/min, 130 ⁇ L/min, 140 ⁇ L/min, 150 ⁇ L/min, or greater.
• the droplet radius may not be dependent on the flow rate of the aqueous fluid 1760 entering the junction 1758 .
• the second fluid 1762 may be stationary, or substantially stationary, in the reservoir 1754 .
• the second fluid 1762 may be flowing, such as at the above flow rates described for the aqueous fluid 1760 .
• FIG. 17 B illustrates the height difference, ⁇ h, being abrupt at the junction 1758 (e.g., a step increase)
• the height difference may increase gradually (e.g., from about 0 ⁇ m to a maximum height difference).
• the height difference may decrease gradually (e.g., taper) from a maximum height difference.
• a gradual increase or decrease in height difference may refer to a continuous incremental increase or decrease in height difference, wherein an angle between any one differential segment of a height profile and an immediately adjacent differential segment of the height profile is greater than 90°.
• a bottom wall of the channel and a bottom wall of the reservoir can meet at an angle greater than 90°.
• a top wall (e.g., ceiling) of the channel and a top wall (e.g., ceiling) of the reservoir can meet an angle greater than 90°.
• a gradual increase or decrease may be linear or non-linear (e.g., exponential, sinusoidal, etc.).
• the height difference may variably increase and/or decrease linearly or non-linearly.
• FIG. 17 B illustrates the expanding reservoir cross-section height as linear (e.g., constant expansion angle, ⁇ )
• the cross-section height may expand non-linearly.
• the reservoir may be defined at least partially by a dome-like (e.g., hemispherical) shape having variable expansion angles.
• the cross-section height may expand in any shape.
• non-barcoded beads can be incorporated into the partitions.
• the biological particle e.g., a cell
• the bead can be a non-barcoded bead.
• a barcode carrying bead can be incorporated into partitions.
• a nucleic acid molecule such as an oligonucleotide
• a releasable linkage such as, for example, a disulfide linker.
• the same bead can be coupled (e.g., via releasable linkage) to one or more other nucleic acid molecules.
• the nucleic acid molecule can be or include a barcode.
• the structure of the barcode can include a number of sequence elements.
• the nucleic acid molecule can include a functional domain that can be used in subsequent processing.
• the functional domain can include one or more of a sequencer specific flow cell attachment sequence (e.g., a P5 sequence for Illumina® sequencing systems (next-generation sequencing)) and a sequencing primer sequence (e.g., a R1 primer for Illumina® sequencing systems (next generation sequencing)).
• the nucleic acid molecule can include a barcode sequence for use in barcoding the sample (e.g., DNA, RNA, protein, etc.). In some cases, the barcode sequence can be bead-specific such that the barcode sequence is common to all nucleic acid molecules coupled to the same bead.
• the barcode sequence can be partition-specific such that the barcode sequence is common to all nucleic acid molecules coupled to one or more beads that are partitioned into the same partition.
• the nucleic acid molecule can include a specific priming sequence, such as an mRNA specific priming sequence (e.g., poly(T) sequence), a targeted priming sequence, and/or a random priming sequence.
• the nucleic acid molecule can include an anchoring sequence to ensure that the specific priming sequence hybridizes at the sequence end (e.g., of the mRNA).
• the anchoring sequence can include a random short sequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or longer sequence, which can ensure that a poly(T) segment is more likely to hybridize at the sequence end of the poly(A) tail of the mRNA.
• the nucleic acid molecule can include a unique molecular identifying sequence (e.g., unique molecular identifier (UMI)).
• UMI unique molecular identifier
• the unique molecular identifying sequence can include from about 5 to about 8 nucleotides. Alternatively, the unique molecular identifying sequence can include less than about 5 or more than about 8 nucleotides.
• the unique molecular identifying sequence can be a unique sequence that varies across individual nucleic acid molecules coupled to a single bead.
• the unique molecular identifying sequence can be a random sequence (e.g., such as a random N-mer sequence).
• the UMI can provide a unique identifier of the starting mRNA molecule that was captured, in order to allow quantitation of the number of original expressed RNA.
• an individual bead can be coupled to any number of individual nucleic acid molecules, for example, from one to tens to hundreds of thousands or even millions of individual nucleic acid molecules.
• the respective barcodes for the individual nucleic acid molecules can include both common sequence segments or relatively common sequence segments and variable or unique sequence segments between different individual nucleic acid molecules coupled to the same bead.
• FIG. 17 C depicts a workflow wherein cells are partitioned into droplets along with barcode-bearing beads 1770 . See FIG. 17 A .
• the droplet forms an isolated reaction chamber wherein the cells can be lysed 1771 and target analytes within the cells can then be captured 1772 and amplified 1773 , 1774 according to previously described methods.
• sequence library preparation clean-up 1775 the material is sequenced and/or quantified 1776 according to methods described herein.
• the poly(T) segment of one of the released nucleic acid molecules can hybridize to the poly(A) tail of a mRNA molecule.
• Reverse transcription can result in a cDNA transcript of the mRNA, which transcript includes each of the sequence segments of the nucleic acid molecule. If the nucleic acid molecule includes an anchoring sequence, it will more likely hybridize to and prime reverse transcription at the sequence end of the poly(A) tail of the mRNA.
• all of the cDNA transcripts of the individual mRNA molecules can include a common barcode sequence segment.
• the transcripts made from the different mRNA molecules within a given partition can vary at the unique molecular identifying sequence segment (e.g., UMI segment).
• UMI segment unique molecular identifying sequence segment
• the number of different UMIs can be indicative of the quantity of mRNA originating from a given partition.
• the transcripts can be amplified, cleaned up and sequenced to identify the sequence of the cDNA transcript of the mRNA, as well as to sequence the barcode segment and the UMI segment.
• nucleic acid molecules bound to the bead can be used to hybridize and capture the mRNA on the solid phase of the bead, for example, in order to facilitate the separation of the RNA from other cell contents.
• precursors that include a functional group that is reactive or capable of being activated such that it becomes reactive can be polymerized with other precursors to generate gel beads that include the activated or activatable functional group.
• the functional group can then be used to attach additional species (e.g., disulfide linkers, primers, other oligonucleotides, etc.) to the gel beads.
• additional species e.g., disulfide linkers, primers, other oligonucleotides, etc.
• some precursors featuring a carboxylic acid (COOH) group can co-polymerize with other precursors to form a bead that also includes a COOH functional group.
• acrylic acid a species comprising free COOH groups
• acrylamide acrylamide
• bis(acryloyl)cystamine can be co-polymerized together to generate a bead with free COOH groups.
• the COOH groups of the bead can be activated (e.g., via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) or 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)) such that they are reactive (e.g., reactive to amine functional groups where EDC/NHS or DMTMM are used for activation).
• EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
• NHS N-Hydroxysuccinimide
• DTMM 4-(4,6-Dimethoxy-1,3,
• the activated COOH groups can then react with an appropriate species (e.g., a species comprising an amine functional group where the carboxylic acid groups are activated to be reactive with an amine functional group) comprising a moiety to be linked to the bead.
• an appropriate species e.g., a species comprising an amine functional group where the carboxylic acid groups are activated to be reactive with an amine functional group
• a degradable bead can be introduced into a partition, such that the bead degrades within the partition and any associated species (e.g., oligonucleotides) are released within the droplet when the appropriate stimulus is applied.
• the free species e.g., oligonucleotides, nucleic acid molecules
• a polyacrylamide bead featuring cystamine and linked, via a disulfide bond, to a barcode sequence can be combined with a reducing agent within a droplet of a water-in-oil emulsion.
• the reducing agent can break the various disulfide bonds, resulting in bead degradation and release of the barcode sequence into the aqueous, inner environment of the droplet.
• heating of a droplet with a bead-bound barcode sequence in basic solution can also result in bead degradation and release of the attached barcode sequence into the aqueous, inner environment of the droplet.
• Any suitable number of species e.g., primer, barcoded oligonucleotide
• the species e.g., primer, e.g., barcoded oligonucleotide
• Such pre-defined concentration can be selected to facilitate certain reactions for generating a sequencing library, e.g., amplification, within the partition.
• the pre-defined concentration of the primer can be limited by the process of producing nucleic acid molecule (e.g., oligonucleotide) bearing beads.
• a degradable bead can include one or more species with a labile bond such that, when the bead/species is exposed to the appropriate stimulus, the bond is broken and the bead degrades.
• the labile bond can be a chemical bond (e.g., covalent bond, ionic bond) or can be another type of physical interaction (e.g., van der Waals interactions, dipole-dipole interactions, etc.)
• a crosslinker used to generate a bead can include a labile bond.
• the labile bond can be broken and the bead degraded.
• a polyacrylamide gel bead that includes cystamine crosslinkers to a reducing agent the disulfide bonds of the cystamine can be broken and the bead degraded.
• a degradable bead can be useful in more quickly releasing an attached species (e.g., a nucleic acid molecule, a barcode sequence, a primer, etc.) from the bead when the appropriate stimulus is applied to the bead as compared to a bead that does not degrade.
• an attached species e.g., a nucleic acid molecule, a barcode sequence, a primer, etc.
• the species can have greater mobility and accessibility to other species in solution upon degradation of the bead.
• a species can also be attached to a degradable bead via a degradable linker (e.g., disulfide linker).
• the degradable linker can respond to the same stimuli as the degradable bead or the two degradable species can respond to different stimuli.
• a barcode sequence can be attached, via a disulfide bond, to a polyacrylamide bead comprising cystamine.
• the bead Upon exposure of the barcoded-bead to a reducing agent, the bead degrades and the barcode sequence is released upon breakage of both the disulfide linkage between the barcode sequence and the bead and the disulfide linkages of the cystamine in the bead.
• degradation can refer to the disassociation of a bound or entrained species from a bead, both with and without structurally degrading the physical bead itself.
• entrained species can be released from beads through osmotic pressure differences due to, for example, changing chemical environments.
• alteration of bead pore sizes due to osmotic pressure differences can generally occur without structural degradation of the bead itself.
• an increase in pore size due to osmotic swelling of a bead can permit the release of entrained species within the bead.
• osmotic shrinking of a bead can cause a bead to better retain an entrained species due to pore size contraction.
• Numerous chemical triggers can be used to trigger the degradation of beads within partitions. Examples of these chemical changes can include, but are not limited to pH-mediated changes to the integrity of a component within the bead, degradation of a component of a bead via cleavage of cross-linked bonds, and depolymerization of a component of a bead.
• a bead can be formed from materials that include degradable chemical cross-linkers, such as BAC or cystamine. Degradation of such degradable cross-linkers can be accomplished through a number of mechanisms.
• a bead can be contacted with a chemical degrading agent that can induce oxidation, reduction or other chemical changes.
• a chemical degrading agent can be a reducing agent, such as dithiothreitol (DTT).
• reducing agents can include ⁇ -mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof.
• a reducing agent can degrade the disulfide bonds formed between gel precursors forming the bead, and thus, degrade the bead.
• a change in pH of a solution can trigger degradation of a bead.
• exposure to an aqueous solution such as water, can trigger hydrolytic degradation, and thus degradation of the bead.
• any combination of stimuli can trigger degradation of a bead.
• a change in pH can enable a chemical agent (e.g., DTT) to become an effective reducing agent.
• Beads can also be induced to release their contents upon the application of a thermal stimulus.
• a change in temperature can cause a variety of changes to a bead. For example, heat can cause a solid bead to liquefy. A change in heat can cause melting of a bead such that a portion of the bead degrades. In other cases, heat can increase the internal pressure of the bead components such that the bead ruptures or explodes. Heat can also act upon heat-sensitive polymers used as materials to construct beads.
• partitions that are formed can include a variety of different reagents and species.
• the lysis reagents can facilitate the release of analytes within the partition.
• lysis agents include bioactive reagents, such as lysis enzymes that are used for lysis of different cell types, e.g., gram positive or negative bacteria, plants, yeast, mammalian, etc., such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and a variety of other lysis enzymes available from, e.g., Sigma-Aldrich, Inc.
• lysis solutions can include non-ionic surfactants such as, for example, TritonX-100 and Tween 20.
• lysis solutions can include ionic surfactants such as, for example, sarcosyl and sodium dodecyl sulfate (SDS).
• Electroporation, thermal, acoustic or mechanical cellular disruption can also be used in certain embodiments, e.g., non-emulsion based partitioning such as encapsulation of analytes that can be in addition to or in place of droplet partitioning, where any pore size of the encapsulate is sufficiently small to retain nucleic acid fragments of a given size, following cellular disruption.
• non-emulsion based partitioning such as encapsulation of analytes that can be in addition to or in place of droplet partitioning, where any pore size of the encapsulate is sufficiently small to retain nucleic acid fragments of a given size, following cellular disruption.
• Examples of other species that can be co-partitioned with analytes in the partitions include, but are not limited to, DNase and RNase inactivating agents or inhibitors, such as proteinase K, chelating agents, such as EDTA, and other reagents employed in removing or otherwise reducing negative activity or impact of different cell lysate components on subsequent processing of nucleic acids. Additional reagents can also be co-partitioned, including endonucleases to fragment DNA, DNA polymerase enzymes and dNTPs used to amplify nucleic acid fragments and to attach the barcode molecular tags to the amplified fragments.
• DNase and RNase inactivating agents or inhibitors such as proteinase K
• chelating agents such as EDTA
• Additional reagents can also be co-partitioned, including endonucleases to fragment DNA, DNA polymerase enzymes and dNTPs used to amplify nucleic acid fragments and to attach the bar
• Additional reagents can also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers and oligonucleotides, and switch oligonucleotides (also referred to herein as “switch oligos” or “template switching oligonucleotides”) which can be used for template switching.
• reverse transcriptase enzymes including enzymes with terminal transferase activity
• primers and oligonucleotides include primers and oligonucleotides, and switch oligonucleotides (also referred to herein as “switch oligos” or “template switching oligonucleotides”) which can be used for template switching.
• switch oligonucleotides also referred to herein as “switch oligos” or “template switching oligonucleotides” which can be used for template switching.
• template switching can be used to increase the length of a cDNA. Template switching can be used
• cDNA can be generated from reverse transcription of a template, e.g., cellular mRNA, where a reverse transcriptase with terminal transferase activity can add additional nucleotides, e.g., poly(C), to the cDNA in a template independent manner.
• Switch oligos can include sequences complementary to the additional nucleotides, e.g., poly(G).
• the additional nucleotides (e.g., poly(C)) on the cDNA can hybridize to the additional nucleotides (e.g., poly(G)) on the switch oligo, whereby the switch oligo can be used by the reverse transcriptase as template to further extend the cDNA.
• Template switching oligonucleotides can include a hybridization region and a template region.
• the hybridization region can include any sequence capable of hybridizing to the target.
• the hybridization region includes a series of G bases to complement the overhanging C bases at the 3′ end of a cDNA molecule.
• the series of G bases can include 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases or more than 5 G bases.
• the template sequence can include any sequence to be incorporated into the cDNA.
• the template region includes at least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequences and/or functional sequences.
• Switch oligos can include deoxyribonucleic acids; ribonucleic acids; bridged nucleic acids, modified nucleic acids including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), inverted dT, 5-Methyl dC, 2′-deoxyInosine, Super T (5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), and combinations of the foregoing.
• 2-Aminopurine 2,6-Diaminopurine
• 2-Amino-dA inverted dT
• beads that are partitioned with the analyte can include different types of oligonucleotides bound to the bead, where the different types of oligonucleotides bind to different types of analytes.
• a bead can include one or more first oligonucleotides (which can be capture probes, for example) that can bind or hybridize to a first type of analyte, such as mRNA for example, and one or more second oligonucleotides (which can be capture probes, for example) that can bind or hybridize to a second type of analyte, such as gDNA for example.
• Partitions can also include lysis agents that aid in releasing nucleic acids from the co-partitioned cell, and can also include an agent (e.g., a reducing agent) that can degrade the bead and/or break covalent linkages between the oligonucleotides and the bead, releasing the oligonucleotides into the partition.
• agent e.g., a reducing agent
• the released barcoded oligonucleotides (which can also be barcoded) can hybridize with mRNA released from the cell and also with gDNA released from the cell.
• Barcoded constructs thus formed from hybridization can include a first type of construct that includes a sequence corresponding to an original barcode sequence from the bead and a sequence corresponding to a transcript from the cell, and a second type of construct that includes a sequence corresponding to the original barcode sequence from the bead and a sequence corresponding to genomic DNA from the cell.
• the barcoded constructs can then be released/removed from the partition and, in some embodiments, further processed to add any additional sequences.
• the resulting constructs can then be sequenced, the sequencing data processed, and the results used to spatially characterize the mRNA and the gDNA from the cell.
• a partition in another example, includes a bead that includes a first type of oligonucleotide (e.g., a first capture probe) with a first barcode sequence, a poly(T) priming sequence that can hybridize with the poly(A) tail of an mRNA transcript, and a UMI barcode sequence that can uniquely identify a given transcript.
• the bead also includes a second type of oligonucleotide (e.g., a second capture probe) with a second barcode sequence, a targeted priming sequence that is capable of specifically hybridizing with a third barcoded oligonucleotide (e.g., an analyte capture agent) coupled to an antibody that is bound to the surface of the partitioned cell.
• the third barcoded oligonucleotide includes a UMI barcode sequence that uniquely identifies the antibody (and thus, the particular cell surface feature to which it is bound).
• the first and second barcoded oligonucleotides include the same spatial barcode sequence (e.g., the first and second barcode sequences are the same), which permits downstream association of barcoded nucleic acids with the partition. In some embodiments, however, the first and second barcode sequences are different.
• the partition also includes lysis agents that aid in releasing nucleic acids from the cell and can also include an agent (e.g., a reducing agent) that can degrade the bead and/or break a covalent linkage between the barcoded oligonucleotides and the bead, releasing them into the partition.
• an agent e.g., a reducing agent
• the first type of released barcoded oligonucleotide can hybridize with mRNA released from the cell and the second type of released barcoded oligonucleotide can hybridize with the third type of barcoded oligonucleotide, forming barcoded constructs.
• the first type of barcoded construct includes a spatial barcode sequence corresponding to the first barcode sequence from the bead and a sequence corresponding to the UMI barcode sequence from the first type of oligonucleotide, which identifies cell transcripts.
• the second type of barcoded construct includes a spatial barcode sequence corresponding to the second barcode sequence from the second type of oligonucleotide, and a UMI barcode sequence corresponding to the third type of oligonucleotide (e.g., the analyte capture agent) and used to identify the cell surface feature.
• the barcoded constructs can then be released/removed from the partition and, in some embodiments, further processed to add any additional sequences.
• the resulting constructs are then sequenced, sequencing data processed, and the results used to characterize the mRNA and cell surface feature of the cell.
• beads that are partitioned can have any of the structures described previously, and can include any of the described combinations of oligonucleotides for analysis of two or more (e.g., three or more, four or more, five or more, six or more, eight or more, ten or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more) different types of analytes within a partition.
• Examples of beads with combinations of different types of oligonucleotides (e.g., capture probes) for concurrently analyzing different combinations of analytes within partitions include, but are not limited to: (a) genomic DNA and cell surface features (e.g., using the analyte capture agents described herein); (b) mRNA and a lineage tracing construct; (c) mRNA and cell methylation status; (d) mRNA and accessible chromatin (e.g., ATAC-seq, DNase-seq, and/or MNase-seq); (e) mRNA and cell surface or intracellular proteins and/or metabolites; (f) a barcoded analyte capture agent (e.g., the MHC multimers described herein) and a V(D)J sequence of an immune cell receptor (e.g., T-cell receptor); and (g) mRNA and a perturbation agent (e.g., a CRISPR crRNA/sgRNA,
• the unaggregated cell or disaggregated cells introduced and processed within partitions or droplets as described herein can be removed from the partition, contacted with a spatial array, and spatially barcoded according to methods described herein.
• single cells of an unaggregated cell sample can be partitioned into partitions or droplets as described herein.
• the partitions or droplets can include reagents to permeabilize a cell, barcode targeted cellular analyte(s) with a cellular barcode, and amplify the barcoded analytes.
• the partitions or droplets can be contacted with any of the spatial arrays described herein.
• the partition can be dissolved, such that the contents of the partition are placed in contact with the capture probes of the spatial array.
• the capture probes of the spatial array can then capture target analytes from the ruptured partitions or the droplets, and processed by the spatial workflows described herein.
• a removal step can optionally be performed to remove all or a portion of the biological sample from the substrate.
• the removal step includes enzymatic and/or chemical degradation of cells of the biological sample.
• the removal step can include treating the biological sample with an enzyme (e.g., a proteinase, e.g., proteinase K) to remove at least a portion of the biological sample from the substrate.
• the removal step can include ablation of the tissue (e.g., laser ablation).
• analyte e.g., detecting the location of an analyte, e.g., a biological analyte
• a biological sample e.g., present in a biological sample
• the method comprising: (a) optionally staining and/or imaging a biological sample on a substrate; (b) permeabilizing (e.g., providing a solution comprising a permeabilization reagent to) the biological sample on the substrate; (c) contacting the biological sample with an array comprising a plurality of capture probes, wherein a capture probe of the plurality captures the biological analyte; and (d) analyzing the captured biological analyte, thereby spatially detecting the biological analyte; wherein the biological sample is fully or partially removed from the substrate.
• a biological sample is not removed from the substrate.
• the biological sample is not removed from the substrate prior to releasing a capture probe (e.g., a capture probe bound to an analyte) from the substrate.
• a capture probe e.g., a capture probe bound to an analyte
• such releasing comprises cleavage of the capture probe from the substrate (e.g., via a cleavage domain).
• such releasing does not comprise releasing the capture probe from the substrate (e.g., a copy of the capture probe bound to an analyte can be made and the copy can be released from the substrate, e.g., via denaturation).
• the biological sample is not removed from the substrate prior to analysis of an analyte bound to a capture probe after it is released from the substrate. In some embodiments, the biological sample remains on the substrate during removal of a capture probe from the substrate and/or analysis of an analyte bound to the capture probe after it is released from the substrate. In some embodiments, analysis of an analyte bound to capture probe from the substrate can be performed without subjecting the biological sample to enzymatic and/or chemical degradation of the cells (e.g., permeabilized cells) or ablation of the tissue (e.g., laser ablation).
• the biological sample is not removed from the substrate prior to analysis of an analyte bound to a capture probe after it is released from the substrate. In some embodiments, the biological sample remains on the substrate during removal of a capture probe from the substrate and/or analysis of an analyte bound to the capture probe after it is released from the substrate. In some embodiments, analysis of an analyte bound to capture probe from the substrate can be performed without subject
• At least a portion of the biological sample is not removed from the substrate.
• a portion of the biological sample can remain on the substrate prior to releasing a capture probe (e.g., a capture prove bound to an analyte) from the substrate and/or analyzing an analyte bound to a capture probe released from the substrate.
• at least a portion of the biological sample is not subjected to enzymatic and/or chemical degradation of the cells (e.g., permeabilized cells) or ablation of the tissue (e.g., laser ablation) prior to analysis of an analyte bound to a capture probe from the substrate.
• analyte e.g., detecting the location of an analyte, e.g., a biological analyte
• a biological sample e.g., present in a biological sample
• permeabilizing e.g., providing a solution comprising a permeabilization reagent to
• the biological sample on the substrate
• contacting the biological sample with an array comprising a plurality of capture probes wherein a capture probe of the plurality captures the biological analyte
• analyzing the captured biological analyte thereby spatially detecting the biological analyte; where the biological sample is not removed from the substrate.
• methods for spatially detecting a biological analyte of interest from a biological sample that include: (a) staining and imaging a biological sample on a substrate; (b) providing a solution comprising a permeabilization reagent to the biological sample on the substrate; (c) contacting the biological sample with an array on a substrate, wherein the array comprises one or more capture probe pluralities thereby allowing the one or more pluralities of capture probes to capture the biological analyte of interest; and (d) analyzing the captured biological analyte, thereby spatially detecting the biological analyte of interest; where the biological sample is not removed from the substrate.
• the method further includes selecting a region of interest in the biological sample to subject to spatial transcriptomic analysis.
• one or more of the one or more capture probes include a capture domain.
• one or more of the one or more capture probe pluralities comprise a unique molecular identifier (UMI).
• UMI unique molecular identifier
• one or more of the one or more capture probe pluralities comprise a cleavage domain.
• the cleavage domain comprises a sequence recognized and cleaved by a uracil-DNA glycosylase, apurinic/apyrimidinic (AP) endonuclease (APE1), U uracil-specific excision reagent (USER), and/or an endonuclease VIII.
• one or more capture probes do not comprise a cleavage domain and is not cleaved from the array.
• a capture probe can be extended.
• extending a capture probe can includes generating cDNA from a captured (hybridized) RNA. This process involves synthesis of a complementary strand of the hybridized nucleic acid, e.g., generating cDNA based on the captured RNA template (the RNA hybridized to the capture domain of the capture probe).
• the captured (hybridized) nucleic acid e.g., RNA
• acts as a template for the extension e.g., reverse transcription, step.
• the capture probe is extended using reverse transcription.
• reverse transcription includes synthesizing cDNA (complementary or copy DNA) from RNA, e.g., (messenger RNA), using a reverse transcriptase.
• reverse transcription is performed while the tissue is still in place, generating an analyte library, where the analyte library includes the spatial barcodes from the adjacent capture probes.
• the capture probe is extended using one or more DNA polymerases.
• a capture domain of a capture probe includes a primer for producing the complementary strand of a nucleic acid hybridized to the capture probe, e.g., a primer for DNA polymerase and/or reverse transcription.
• the nucleic acid, e.g., DNA and/or cDNA, molecules generated by the extension reaction incorporate the sequence of the capture probe.
• the extension of the capture probe e.g., a DNA polymerase and/or reverse transcription reaction, can be performed using a variety of suitable enzymes and protocols.
• a full-length DNA, e.g. cDNA, molecule is generated.
• a “full-length” DNA molecule refers to the whole of the captured nucleic acid molecule. However, if the nucleic acid, e.g. RNA, was partially degraded in the tissue sample, then the captured nucleic acid molecules will not be the same length as the initial RNA in the tissue sample.
• the 3′ end of the extended probes, e.g., first strand cDNA molecules is modified. For example, a linker or adaptor can be ligated to the 3′ end of the extended probes.
• RNA ligase a single stranded ligation enzyme
• template switching oligonucleotides are used to extend cDNA in order to generate a full-length cDNA (or as close to a full-length cDNA as possible).
• a second strand synthesis helper probe (a partially double stranded DNA molecule capable of hybridizing to the 3′ end of the extended capture probe), can be ligated to the 3′ end of the extended probe, e.g., first strand cDNA, molecule using a double stranded ligation enzyme such as T4 DNA ligase.
• a polynucleotide tail e.g., a poly(A) tail, is incorporated at the 3′ end of the extended probe molecules. In some embodiments, the polynucleotide tail is incorporated using a terminal transferase active enzyme.
• double-stranded extended capture probes are treated to remove any unextended capture probes prior to amplification and/or analysis, e.g. sequence analysis. This can be achieved by a variety of methods, e.g., using an enzyme to degrade the unextended probes, such as an exonuclease enzyme, or purification columns.
• extended capture probes are amplified to yield quantities that are sufficient for analysis, e.g., via DNA sequencing.
• the first strand of the extended capture probes e.g., DNA and/or cDNA molecules
• acts as a template for the amplification reaction e.g., a polymerase chain reaction.
• the amplification reaction incorporates an affinity group onto the extended capture probe (e.g., RNA-cDNA hybrid) using a primer including the affinity group.
• the primer includes an affinity group and the extended capture probes includes the affinity group.
• the affinity group can correspond to any of the affinity groups described previously.
• the extended capture probes including the affinity group can be coupled to a substrate specific for the affinity group.
• the substrate can include an antibody or antibody fragment.
• the substrate includes avidin or streptavidin and the affinity group includes biotin.
• the substrate includes maltose and the affinity group includes maltose-binding protein.
• the substrate includes maltose-binding protein and the affinity group includes maltose.
• amplifying the extended capture probes can function to release the extended probes from the surface of the substrate, insofar as copies of the extended probes are not immobilized on the substrate.
• the extended capture probe or complement or amplicon thereof is released.
• the step of releasing the extended capture probe or complement or amplicon thereof from the surface of the substrate can be achieved in a number of ways.
• an extended capture probe or a complement thereof is released from the array by nucleic acid cleavage and/or by denaturation (e.g. by heating to denature a double-stranded molecule).
• the extended capture probe or complement or amplicon thereof is released from the surface of the substrate (e.g., array) by physical means.
• the extended capture probe is indirectly immobilized on the array substrate, e.g. via hybridization to a surface probe, it can be sufficient to disrupt the interaction between the extended capture probe and the surface probe.
• Methods for disrupting the interaction between nucleic acid molecules include denaturing double stranded nucleic acid molecules art.
• a straightforward method for releasing the DNA molecules i.e., of stripping the array of the extended probes
• the extended capture probe is released by applying heated water such as water or buffer of at least 85° C., e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99° C.
• heated water such as water or buffer of at least 85° C.
• a solution including salts, surfactants, etc. that can further destabilize the interaction between the nucleic acid molecules is added to release the extended capture probe from the substrate.
• the extended capture probe is released from the surface of the substrate by cleavage.
• the cleavage domain of the extended capture probe can be cleaved by any of the methods described herein.
• the extended capture probe is released from the surface of the substrate, e.g., via cleavage of a cleavage domain in the extended capture probe, prior to the step of amplifying the extended capture probe.
• Capture probes can optionally include a “cleavage domain,” where one or more segments or regions of the capture probe (e.g., spatial barcodes and/or UMIs) can be releasably, cleavably, or reversibly attached to a feature, or some other substrate, so that spatial barcodes and/or UMIs can be released or be releasable through cleavage of a linkage between the capture probe and the feature, or released through degradation of the underlying support, allowing the spatial barcode(s) and/or UMI(s) of the cleaved capture probe to be accessed or be accessible by other reagents, or both.
• cleavage domain where one or more segments or regions of the capture probe (e.g., spatial barcodes and/or UMIs) can be releasably, cleavably, or reversibly attached to a feature, or some other substrate, so that spatial barcodes and/or UMIs can be released or be re
• the capture probe is linked, via a disulfide bond, to a feature.
• the capture probe is linked to a feature via a propylene group (e.g., Spacer C3).
• a reducing agent can be added to break the various disulfide bonds, resulting in release of the capture probe including the spatial barcode sequence.
• heating can also result in degradation and release of the attached capture probe.
• the heating is done by laser (e.g., laser ablation) and features at specific locations can be degraded.
• labile bonds that can be coupled to a capture probe (i.e., spatial barcode) include an ester linkage (e.g., cleavable with an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g., cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavable via heat), a sulfone linkage (e.g., cleavable via a base), a silyl ether linkage (e.g., cleavable via an acid), a glycosidic linkage (e.g., cleavable via an amylase), a peptide linkage (e.g., cleavable via a protease), or a phosphodiester linkage (e.g.,